Appendix 3 of Singh, Mol Vis 2016; 22:1036-1047.
To access the data, click or select the words “Appendix 3.” Detection of copy number variation (CNV) and confirmation by MLPA (multiplex ligation-dependent probe amplification). Figures on the left panel (A, C, E, G, I and K) represent CNV analysis and on the right panel (B, D, F, H and J) represent MLPA analysis. In the MLPA plot, x-axis represents genomic regions and y-axis represents dosage quotient (DQ). DQ distribution of 0.8–1.2 represent normal copy, 0.4–0.65 represents heterozygous deletion and 1.3–1.65 represents heterozygous duplication. In the sample RB6, the CNV analysis showed a heterozygous deletion of exon 8–11 (A), which was confirmed by MLPA analysis (B). Similarly, the CNV analysis in the sample RB30, revealed a heterozygous deletion of exon 25 (C), which was confirmed by MLPA analysis (D). The CNV analysis in the sample RB31, revealed a heterozygous deletion of exon 26–27 and a partial deletion of exon 25 (depicted in dotted circle; E), which was confirmed by MLPA analysis (F). The CNV analysis in the sample RB32 showed heterozygous whole RB1 gene deletion (G), which was confirmed by MLPA analysis (H). The CNV analysis in the sample RB33 showed heterozygous whole RB1 gene deletion (I); although the CNV data showed higher heterogeneity, however, this deletion was confirmed by MLPA analysis (J). In both cases of whole gene deletion (RB32 and RB33), MLPA analysis revealed that in addition to the whole RB1 gene deletion, the upstream and downstream genomics regions flanking the RB1 gene were also deleted. The CNV analysis in the sample RB29, revealed a heterozygous deletion of exon 24–27, this sample also showed higher heterogeneity in the CNV data compared to other samples; however, considering deletion of multiple continuous exons, it is unlikely that it is false positive (K), as, additional DNA was unavailable for the sample RB29 therefore MLPA confirmation could not be performed.