Figure legends:
Figure 1. Pedigree showing segregation of a 145 Kbp duplication at 18q21.1, including exons 1-6 of SMAD2 . Proband is indicated with a black arrow (D4).
Figure 2. arr[GRCh37] 18q21.1(45384387_45529362)x3 mat duplication in proband. A) Screenshot of the duplication position as seen in ChAS software. The duplication is shown in the first horizontal panel as a blue rectangle. Probe positions are shown in the middle panel as dots. Notice the elevated signal for probes directly below the blue rectangle, which were used for calling the duplication positions. Gene positions (SMAD2 and ZBTB7C ) are shown in the third panel, along with the nucleotide positions in chromosome 18. Exon numbers are displayed for SMAD2 . The shaded rectangle encompassesSMAD2 positions, and shows the partial duplication. The 145 Kbp duplication overlapped exons one through six of SMAD2 , which could potentially disrupt the gene’s function. In addition, the duplication was found to be maternally inherited in proband, but the mother was phenotypically normal. B) The SMAD2 rearrangement as seen in our MPseq analysis pipeline. This junction plot displays four breakpoints representing the inversion event (colored dots indicate orientation of mapped reads, with blue corresponding to forward strand and red the reverse strand). Notice the presence of the blue-blue and red-red dot associations predicted to be seen in a duplication with inverted orientation. Only discordant mate-pair reads with end positions mapping >15 kb are shown. C) Schematic representation of the SMAD2 duplication seen in proband compared to the reference allele. Concordant and discordant mate-pair fragments and their corresponding orientations are shown in colored text.
Figure 3. SMAD2 RNA expression analysis. Total mRNA was isolated from cultured proband amniocytes, and RNA extracted from the amniocytes of a normal male with normal SMAD2 copy number and no phenotypic evidence of dextrocardia was used as a control. Real-time RT-PCR analysis targeted the SMAD2 exon junctions 2/3, 4/5, 6/7, and 9/10 indicated underneath the black horizontal bars in both the proband (grey bars and Pt label) and the control (black bars and Ctrl label). NTC: no template control. The expression of SMAD2 is significantly reduced in proband compared to the normal control.