Copy Number Variations Due to Large Genomic Deletion in X-Linked Chronic Granulomatous Disease

Mutations in genes for any of the six subunits of NADPH oxidase cause chronic granulomatous disease (CGD), but almost 2/3 of CGD cases are caused by mutations in the X-linked CYBB gene, also known as NAD (P) H oxidase 2. Approximately 260 patients with CGD have been reported in Japan, of whom 92 were shown to have mutations of the CYBB gene and 16 to have chromosomal deletions. However, there has been very little detailed analysis of the range of the deletion or close understanding of the disease based on this. We therefore analyzed genomic rearrangements in X-linked CGD using array comparative genomic hybridization analysis, revealing the extent and the types of the deletion genes. The subjects were five Japanese X-linked CGD patients estimated to have large base deletions of 1 kb or more in the CYBB gene (four male patients, one female patient) and the mothers of four of those patients. The five Japanese patients were found to range from a patient exhibiting deletions only of the CYBB gene to a female patient exhibiting an extensive DNA deletion and the DMD and CGD phenotype manifested. Of the other three patients, two exhibited CYBB, XK, and DYNLT3 gene deletions. The remaining patient exhibited both a deletion encompassing DNA subsequent to the CYBB region following intron 2 and the DYNLT3 gene and a complex copy number variation involving the insertion of an inverted duplication of a region from the centromere side of DYNLT3 into the deleted region

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

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 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

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

Clinical Courses of IKAROS and CTLA4 Deficiencies: A Systematic Literature Review and Retrospective Longitudinal Study

International audienceIKAROS and CTLA4 deficiencies are inborn errors of immunity and show similar clinical phenotypes, including hypogammaglobulinemia and autoimmune diseases (ADs). However, the differences in clinical features and pathogenesis of these are not fully understood. Therefore, we performed systematic literature reviews for IKAROS and CTLA4 deficiencies. The reviews suggested that patients with IKAROS deficiency develop AD earlier than hypogammaglobulinemia. However, no study assessed the detailed changes in clinical manifestations over time; this was likely due to the cross-sectional nature of the studies. Therefore, we conducted a retrospective longitudinal study on IKAROS and CTLA4 deficiencies in our cohort to evaluate the clinical course over time. In patients with IKAROS deficiency, AD and hypogammaglobulinemia often develop in that order, and AD often resolves before the onset of hypogammaglobulinemia; these observations were not found in patients with CTLA4 deficiency. Understanding this difference in the clinical course helps in the clinical management of both. Furthermore, our results suggest B- and T-cell-mediated ADs in patients with IKAROS and CTLA4 deficiencies, respectively