PMC

In approximately 90% of the patients with the DiGeorge syndrome, the congenital disorder is caused by a classic de novo heterozygous deletion of approximately 2.5 mb in length spanning chromosome 22q11.2 low-copy repeats (LCR22) A and D, as shown in blue. Less than 10% of the patients with this syndrome carry the critical 1.5-mb deletion between LCR22 A and B. Shown in red are deletions that were identified in 14 patients who were affected by congenital anomalies of the kidney and urinary tract among the 2080 patients who were tested. According to the megabase coordinates for the Human Genome 19 release, the proximal and distal breakpoints for the chromosome 22q11.2 deletions that were identified in the patients are as follows: P1, 18.88 to 21.47 mb; P2, 18.89 to 21.47 mb; P3, 20.73 to 21.46 mb; P4, 21.02 to 22.47 mb; P5, 21.05 to 21.47 mb; P6, 21.06 to 21.47 mb; P7, 21.06 to 21.46 mb; P8, 21.06 to 21.46 mb; P9, 21.07 to 21.46 mb; P10, 21.08 to 21.47 mb; P11, 21.09 to 21.47 mb; Patient 1 from the replication cohort (RP1), 18.88 to 21.46 mb; RP2, 20.74 to 21.46 mb; and RP3, 20.74 to 21.46 mb. The deletion between LCR22 C and D defines the smallest region of overlap for congenital kidney disease among patients with 22q11.2 deletions.

Panel A shows immunostaining for Crkl in kidney obtained from a transgenic mouse on embryonic day E15.5, in which Six2 has been tagged with enhanced green fluorescent protein (GFP), with specific Crkl staining of the ureteric bud (in red) surrounded by Six2-positive cap mesenchyme cells (in green) (subpanel a). A magnified field shows ureteric-bud branching within condensing metanephric mesenchyme (subpanel b). Panel B shows specific pronephros expression of crkl in zebrafish, as shown by colocalization after staining with antibody against sodium–potassium ATPase. In the orientation symbol, D denotes dorsal, V ventral, C caudal, and R rostral. Panel C shows images of negative controls (i.e., fish treated with fluorophore-conjugated secondary antibodies only). In Panels B and C, the scale bars represent 100 μm. In a mouse model that targets Crkl exon 2, three crosses with transgenic Cre-recombinase mice were created to effect the deletion of exon 2 in specific compartments: E2a-Cre for global knockout, Six2-Cre in the cap mesenchyme, and Hoxb7 in the structures derived from ureteric buds. Panel D shows tissue from a Six2-Cre mouse in which duplication of the right kidney is accompanied by an irregular, dysplastic pattern or ureteric-bud branching on embryonic day E15.5. Panel E shows tissue from an E2a-Cre mouse in which a single kidney with duplicated ureters (arrowheads) is accompanied by failure of medullary and renal papillary development on day E14.5. Panel F shows tissue from a Six2-Cre mouse, in which the kidney is hydronephrotic with dilated pelvis, absence of medullary architecture, and several microcystic glomeruli and tubules on day E15.5.

Panel A shows zebrafish larvae 4.5 days after fertilization, in which the proximal tubule is folded into a hairpin structure, displaying proper anterior convolution in noninjected control embryos (staining with antibody against sodium–potassium ATPase). Knockdown of ret, aifm3, crkl, and snap29 by the injection of 8.0 ng of a splice-blocking morpholino oligonucleotide (MO) against RET resulted in major convolution defects, which are apparent by the failure of the anterior portion of the pronephros (the earliest developmental stage in the zebrafish) to progress, along with an overall reduction in the length of the tubules. Panel B shows the relative length of the pronephros, which was defined as the ratio of the length of the pronephros (a) to the length of the body axis (b), in individual larvae (inset). The number of replicate measurements were as follows: control or sham-injected control, 177 in Panel A and 68 in Panel B; ret-MO, 50; ret-MO+mRNA, 42; aifm3-MO, 38; aifm3-MO+mRNA, 42; crkl-MO, 43; crkl-MO+mRNA, 58; snap29-MO, 48; snap29-MO+mRNA, 39; ret-gRNA+Cas9, 44; crkl-gRNA+Cas9, 31; and snap29-gRNA+Cas9, 41). Morphant phenotypes could be rescued by the coinjection of each respective human messenger RNA (mRNA). In each box-and-whisker plot, the horizontal line represents the median, the top and bottom of the boxes the interquartile range, and the I bars the minimum and maximum values. Panel C shows embryos that have been injected with CRISPR–Cas9 and that are reproducing the convolution defects observed in the morphant embryos. Guide RNA (gRNA) that targeted each respective gene was coinjected with purified Cas9 protein, and the relative length of the pronephros was measured in founders, as shown in Panel D. In Panels B and D, a single asterisk indicates P<0.05, two asterisks P<0.01, and three asterisks P<0.001. WT denotes wild type.