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.