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. 2016 Sep 15;99(4):831–845. doi: 10.1016/j.ajhg.2016.08.007

Figure 3.

Figure 3

Compound Heterozygous Copy-Number Variation in Family 7

(A) Miropeats alignment of the ATAD3 region indicates homology of the three ATAD3 paralogs. ATAD3C reference sequence is indicated in red, ATAD3B reference sequence is indicated in green, and ATAD3A reference sequence is indicated in blue.

(B) Target Z score of PCA-normalized read depths for 44 exon targets of the three ATAD3 genes. Comparative read depth data obtained from 2,634 WES samples are shown in gray (clinical collection). Family 7, II-1 is shown in red. Target Z score of PCA-normalized read depths for 16 exons (x axis) spanning portions of ATAD3B and ATAD3A have large negative Z scores (y axis), indicating a copy loss of both alleles.

(C) Droplet Digital PCR (ddPCR) detected a larger heterozygous deletion in BAB8734 (father) than the heterozygous deletion in BAB8733 (mother). Primer pairs targeting various exonic or intronic regions of ATAD3C, ATAD3B, and ATAD3A and a control primer pair targeting RPPH1 were used to perform ddPCR in BAB8733 (mother), BAB8734 (father), and a control individual N/A10851. RPPH1 is a gene known to have exactly two copies in the human genome. Ratios of concentrations of positive droplets were plotted for each amplicon tested against the control individual. For a region without deletion, the expected ratio is ∼1, whereas for heterozygous deletion, the expected ratio is ∼0.5 (one deleted copy-number versus two copies). Corresponding raw data of ddPCR and primer sequences are shown in Table S1.

(D) Schematic of primer design targeting maternal and paternal deletions and wild-type alleles. PCR amplicons indicating segregation of maternal deletion allele (F1/R1) and paternal deletion allele (F3/R3). Note that proband (BAB8780) lacks wild-type allele amplified by F2/R1 but does have wild-type allele amplified by F3/R4 since the latter is positioned outside the maternal deletion and can be amplified from this allele.

(E) Results of PCR breakpoint analysis. Proband inherited both the maternal and paternal deletions.