Introduction
Accurate characterization of genomic structural variation is essential
to prenatal genetic screening and clinical diagnosis of fetuses with
abnormal ultrasound findings. Currently, prenatal genomic abnormalities
ranging from aneuploidy to single-nucleotide variants (SNVs) can be
detected with a combination of genetic testing strategies, including but
not limited to chromosome karyotyping, fluorescence in-situ
hybridization (FISH), chromosome microarray (CMA), and next-generation
sequencing (NGS) [1-4]. CMA in particular has been shown to
substantially increase prenatal diagnostic yields compared to chromosome
banding analyses, given its ability to detect clinically relevant
submicroscopic duplications and deletions (copy-number variants, CNVs)
[1, 5]. Chromosome deletions have been extensively associated with
abnormal phenotypes through their impact on haploinsufficient gene
expression, an observation that facilitates prenatal deletion
classification and reporting. In contrast, fetal chromosome duplications
have posed a more challenging and potentially uncertain interpretative
context: at the functional level, duplications have the potential to
result in overexpression of triplosensitive genes, disruption of
haploinsufficient genes at their breakpoints, or creation of gene
fusions, all of which could contribute to pathogenesis [6]. The
clinical utility of characterizing duplications by NGS to inform their
mechanism of pathogenesis has been previously shown [6], with
duplications in inverted orientation being more often associated with
complex chromosome rearrangements.
Mate pair sequencing (MPseq) is an NGS technology specifically designed
for the detection of genomic structural variants [7, 8]. The method
is based on the generation of large insert libraries (2-5kb) followed by
paired-end (PE) sequencing. Mapping of PE reads can easily reveal the
presence of structural rearrangements with a high degree of confidence,
including translocations, inversions, insertions, and copy-number
variants [8-10].
In this report, we describe the MPseq analysis of a fetus with
dextrocardia, pulmonary hypoplasia, and a complex conotruncal anomaly.
Dextrocardia is a rare congenital condition in which the heart points
towards the right side of the chest rather than the left [11] and is
frequently associated with heterotaxy, with important morbidity
contributions due to abnormal pulmonary venous connection and ventricle
obstructions [12]. CMA performed on fetal amniotic fluid revealed a
145 Kbp duplication at 18q21.1, including exons 1-6 of the SMAD Family
Member 2 (SMAD2 ) gene. While SMAD2 plays an important role
in mouse left-right embryonic patterning [13] and mutations have
been linked to dextrocardia [14] and other heart abnormalities in
humans [15], the CMA results alone could not be used to determine if
the duplicated segment disrupted SMAD2 gene function. MPseq was
necessary to clarify the role of this duplication in the clinical
phenotype of the fetus by revealing the duplicated segment to be in
inverted tandem orientation to the normal chromosome segment. This
finding was predicted to disrupt SMAD2 as evidenced by its
reduced gene expression in cultured amniocytes.