Genomic Rearrangement Mechanisms.

<p>For the gene deletions in patient 1, gene rearrangements exhibiting deletions due to non-homologous end-joining (NHEJ) were thought to have occurred, since repeated two base sequences of TA were observed at both ends of the gene (1a, 1b). In patient 2, it was hypothesized that rearrangement might have occurred through a mechanism involving a combination of fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR). A three base repeat sequence of AAT prone to cause changes in copy number or sequence swaps was observed in a discontinuous end of the lagging strand during DNA replication. This formed a loop by binding to TTA on its complementary strand with replication slippage occurring (2a). At the discontinuous end, the lagging strand with the loop formed had a six base microhomology of AGGTAT and dissociated. The dissociated end subsequently bound to the ATACCT found at the duplication front of the leading strand. Synthesis and extension of the leading strand then occurred originating from the binding site (2b). The gene synthesized on the leading strand is hypothesized to cause structural abnormalities leading to duplications of the DNA strand subsequent to the DNA strand deletion by returning to the lagging strand after the end of duplication and the binding of the end that has finished duplication to the starting end of the normal gene on the lagging strand by DNA ligase (2c).</p

The Results of aCGH and Direct Sequence Analysis Spanning the Deletion Breakpoints in Patient 2.

<p>A female's DNA was used for the control. According to aCGH, the deletion site was 84.4 kbp and the duplication site 91.9 kb. According to the DNA walking analysis by PCR, a breakpoint was located at 37398670 of <i>CYBB</i> intron 2. Furthermore, six bases of the gene at 37612392 (UCSC hg17 May.2004) of ATACCT were bound inverted at the breakpoint, and complex structural abnormalities showing gene deletions on the inner side and duplication of the inverted part were observed.</p

The Results of aCGH Analysis Spanning the Deletion Breakpoints in Patients 3 and 4.

<p>A female's DNA was used for the control DNA for patient 3, and a male's for patient 4. These male infants suffered deletions of three genes: the chronic granulomatous disease gene (<i>CYBB</i>), the McLeod syndrome gene (<i>XK</i>), and <i>DYNLT3</i>. The gene deletions in each patient were found to be 0.59 Mb and 1.94 Mb, respectively.</p

The Results of aCGH Analysis Spanning the Deletion Breakpoints in Mother and Patient 5.

<p>A female's DNA was used for the control DNA. This case involved deletions of four genes, the chronic granulomatous disease gene (<i>CYBB</i>), the Duchenne muscular dystrophy gene (<i>DMD</i>), the McLeod syndrome gene (<i>XK</i>), and <i>DYNLT3</i>. The gene deletion region was about 5.71 Mb and encompassed an area from <i>CYBB</i> to the greater part of the <i>DMD</i> (b). A similar gene deletion was also observed in the mother (a).</p

PCR Amplification Results for Exons 1 and 13 of the Chronic Granulomatous Disease Gene <i>CYBB</i> and Exon 1 of the McLeod Syndrome Gene <i>XK</i> Region.

<p>The analysis used the Agilent 2100 Bioanalyzer. Patients 1 to 5 were juvenile patients with chronic granulomatous disease, and genes from normal boys were amplified as a control. <i>CYBB</i> exon 1 was detected only from patients 2 and 5, and <i>CYBB</i> exon 13 only from patient 5. In addition, <i>XK</i> exon 1 was detected in patients 1, 2 and 5.</p