Fig. 3

Long-Range PCR of the putative POLR3B exonic deletions and sequencing of the breakpoints. a. Patient 1: Long-Range (LR- PCR) was performed on genomic DNA using exon-specific forward and reverse primers for exons 20 and 23 respectively. The expected fragment size of this region was estimated at 10 kb (Control DNA; Lane 2 and U51.2; Lane 5), however the proband LR-PCR amplification generated a smaller DNA fragment with average product length of approximately 3 kb, as indicated by the arrow (U51.0; Lane 3). This 3 kb DNA fragment was also seen in the LR-PCR amplification of paternal DNA (U51.1; Lane 4), but not of maternal DNA (U51.2; Lane 5). Lane 1 is the molecular size marker. Sanger sequencing of the abnormal fragment identified in both the proband and the unaffected father allowed mapping of the breakpoint on the 5’ at position 106, 751, 658 on chromosome 12, and the breakpoint at the 3’ was at position 106, 857, 267 (data not shown). No homology was found at either of the deletion breakpoints. Tandem Repeats finder, QuadParser and Repeat Masker databases were used for sequence motif analysis. These tools failed to identify motif, suggesting that homologous recombination events were unlikely. The exonic deletions of POLR3B appear to have arisen by the simple rejoining of non-homologous DNA ends during double stranded break repair. b. Patient 2: Long-Range (LR- PCR) was performed on genomic DNA using intron-specific forward and reverse primers for introns 25 and 27 respectively. The expected fragment size of this region was estimated at 13 kb (Control DNA; Lane 3), however the proband LR-PCR amplification generated a smaller DNA fragment with average product length of approximately 9 kb, as indicated by the arrow (08D1250; Lane 2). Lane 1 and 4 is the molecular size marker