Journal: medRxiv

Article Title: Single-exon deletions of ZNRF3 exon 2 cause congenital adrenal hypoplasia

doi: 10.1101/2023.06.14.23290643

Figure Lengend Snippet: Identification of exon 2 deletions in ZNRF3 in three PAI patients using CNV analyses and RT-PCR. A. Schematic diagram of full-length ZNRF3 transcript. Blue and red areas represent translated region, gray untranslated region, and red exon 2. The facing arrowheads indicate primer sites for RT-PCR. B. Array CGH plots for P1-3, who have various-sized deletions encompassing exon 2 in ZNRF3 . On the left panel, blue line indicates the region of interest ( ZNRF3 ) in an idiogram of 22q11.2-22q13.1. Green-shaded region decreased in copy number, and unshaded region had normal copy number. Right panel shows the gene structure of ZNRF3 . C. FISH study in P1 and P2, who have exon 2 deletions in ZNRF3 . Both patients have one red signal (arrow) on the region of ZNRF3 exon 2 on Chr22, and two green control signals (arrow heads) on a pair of Chr22. D. Agarose-gel electrophoresis of RT-PCR amplicons using the primers spanning ZNRF3 exons 1 to 5 . P1 and P2 have two fragments of 759- and 633-bp, and control (C) has the only 759-nt fragment. E. Sequence chromatograms of the obtained RT-PCR amplicons from P1 and P2. P1 and P2 have both a wild-type allele and a mutant allele skipping exon 2 sequence. F. Pedigrees of three families with exon 2 deletions in ZNRF3 . Solid symbols denote affected individuals with neonatal-onset PAI, open symbols unaffected ones. WT and ΔEx2 denote wild-type and exon 2 deletion, respectively. Maternal mosaicism of ΔEx2 was identified in P2 family.

Article Snippet: Two guide RNAs (gRNAs) sequences that flank ZNRF3 exon 2 (Figure S2A) were cloned into pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro (Addgene #42229).

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing, Mutagenesis

Journal: medRxiv

Article Title: Single-exon deletions of ZNRF3 exon 2 cause congenital adrenal hypoplasia

doi: 10.1101/2023.06.14.23290643

Figure Lengend Snippet:

Article Snippet: Two guide RNAs (gRNAs) sequences that flank ZNRF3 exon 2 (Figure S2A) were cloned into pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro (Addgene #42229).

Techniques:

Journal: medRxiv

Article Title: Single-exon deletions of ZNRF3 exon 2 cause congenital adrenal hypoplasia

doi: 10.1101/2023.06.14.23290643

Figure Lengend Snippet: Functional characterization of exon 2 deletion in ZNRF3 . A. 3D structure of Znrf3-RSPO1 complex. Transparent surface representation of the extracellular region of Znrf3 (pink; Exon 1,3 and 4, red; Exon 2) and RSPO1 (green). Based on the two views, exon 2 of Znrf3 makes a contact to RSPO1. B. ZNRF3 protein expression analyzed by means of Western blotting. WT and ΔEx2 protein expression in transfected HEK293 cells are comparable. C. Intracellular localization by immunofluorescence staining of transfected HEK293 cells with WT and ΔEx2 constructs. WT and ΔEx2 FLAG-tagged ZNRF3 (red) are localized to the plasma membrane. DAPI (blue) shows cell nuclei. D. The relative effect of each ZNRF3 protein (WT, ΔEx2, or H102A/P103A) on Wnt/dependent β-catenin signaling, as assessed by means of a luc2P/TCF-LEF-reporter assay. H102A/P103A denotes the previously described constitutive active artificial variant of p.His102Ala;Pro103Ala (34). The luciferase activity under RSPO1 and Wnt3a treatment was lower in cells expressing ΔEx2, as well as of H102A/P103A mutant, than those expressing WT. Data are representative of three independent experiments. E. Expression of Znrf3, Shh and Nr5a1 in adrenal cortex of fetal mice (E16.5) by means of in situ hybridization (ISH). Upper panels show ISH with antisense probes, and lower panels ISH with sense probes. Znrf3 , as well as Shh , mainly expressed in subcapsular cells of the adrenal cortex, while Nr5a1 was expressed throughout the adrenal cortex.

Article Snippet: Two guide RNAs (gRNAs) sequences that flank ZNRF3 exon 2 (Figure S2A) were cloned into pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro (Addgene #42229).

Techniques: Functional Assay, Expressing, Western Blot, Transfection, Immunofluorescence, Staining, Construct, Reporter Assay, Variant Assay, Luciferase, Activity Assay, Mutagenesis, In Situ Hybridization