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Dominant human genetic diseases that impair reproductive fitness and have high locus heterogeneity constitute a problem for gene discovery because the usual criterion of finding more mutations in specific genes than expected by chance may require extremely large populations. Heterotaxy (Htx), a congenital heart disease resulting from abnormalities in left-right (LR) body patterning, has features suggesting that many cases fall into this category. In this setting, appropriate model systems may provide a means to support implication of specific genes. By high-resolution genotyping of 262 Htx subjects and 991 controls, we identify a twofold excess of subjects with rare genic copy number variations in Htx (14.5% vs. 7.4%, P = 1.5 × 10(-4)). Although 7 of 45 Htx copy number variations were large chromosomal abnormalities, 38 smaller copy number variations altered a total of 61 genes, 22 of which had Xenopus orthologs. In situ hybridization identified 7 of these 22 genes with expression in the ciliated LRorganizer (gastrocoel roof plate), a marked enrichment compared with 40 of 845 previously studied genes (sevenfold enrichment, P < 10(-6)). Morpholino knockdown in Xenopus of Htx candidates demonstrated that five (NEK2, ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both morphological LR development and expression of pitx2, a molecular marker of LR patterning. These effects were specific, because 0 of 13 control genes from rare Htx or control copy number variations produced significant LR abnormalities (P = 0.001). These findings identify genes not previously implicated in LR patterning.
Fig. 3. WMISH analysis. (A–U) Results of in situ hybridization at three stages are shown for seven genes showing patterns of interest. These genes show expression in one or more of the following: GRP (blue arrows); heart or branchial arches (red arrows); kidney (green arrows). Stage 15 to 19 embryos are viewed dorso-posteriorly with anterior to the top to examine GRP expression (shown schematically in V). Stage 26 to 29 and stage 33 to 36 embryos are viewed laterally with anterior to the left (shown schematically in W and X, respectively).
Fig. 5. Analysis of pitx2 expression in stage 28 to 30 Xenopus embryos. Embryos are viewed laterally from the left (first column) and the right (second column). Note normal, bilateral pitx2 expression in the head region in all embryos. (A) Expression of pitx2 is normally in the leftlateral plate mesoderm (LPM, arrow). (B) Same normal embryo showing absent pitx2 expression in the rightLPM. (C and D) Absent pitx2 expression. No pitx2 mRNA is found in the left or rightLPM. (E and F) Bilateral pitx2 expression. The pitx2 mRNA is found in both left and rightLPM (arrows). (G and H) Rightpitx2 expression: pitx2 mRNA is absent from the leftLPM, present in the rightLPM. (Graph) Summary of pitx2 mRNA expression in MO knockdown embryos: dnah9 is a positive control; StdCtrl, and UiC are negative controls. Bars show the percent of abnormal pitx2 expression and are divided into blue (bilateral), red (right), and green (absent) LPMpitx2 expression.
greb1 (growth regulation by estrogen in breast cancer 1) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anteriorleft, dorsal up.
LR abnormalities from MO knockdown in X. tropicalis. MOs were injected at the one-cell stage and heart and gut looping were assayed in tadpoles at stage 45/46. Views are from the ventral aspect, shown in schematic form in F. (A) Heart (area outlined in red box as in schematic in F) showing normal D-looping. The inflow (red arrow) is on the tadpole's left, the outflow tract (yellow arrow) is on the tadpole's right. (B) Heart showing abnormal, anterior, A-looping. The inflow (red) and outflow (yellow) are both at the midline, with no discernible LR orientation. (C) Heart showing abnormal, reversed, L-looping. The inflow (red) is on the tadpole's right, the outflow (yellow) is on the tadpole's left. (D) Normal clockwise rotation of the gut. (E) Abnormal gut rotation. (F) Schematic of Xenopus tadpole at stage 45/46; ventral view with anterior to the top; arrows indicate heart and gut. (G) Heart looping in MO knockdown tadpoles. Both dnah9 and ift88 are positive controls; standard control MO (StdCtrl), uninjected control (UiC), and dye-injected (Dye) are negative controls. Bars show the total percentage of abnormally looped hearts: divided into A-loop (blue) and l-loop (red). (H) Gut looping in MO knockdown tadpoles. Both dnah9 and ift88 MOs are used as positive controls; StdCtrl, UiC, and Dye are used as negative controls. Red bars show the the percent of abnormal gut loops. Heart and gut looping were analyzed by two independent readers blinded to group status with 95% concordance. *P < 10−4 vs. StdCtrl.
Fig. 2. Rare genic deletions and duplications in Htx patients. Results of Illumina genotyping and qPCR are shown for four CNVs at three loci that contain genes implicated in Htx. In all panels, genes in the indicated chromosome segment are shown and genes implicated in Htx are denoted by arrows. Data from subjects with deletion or duplications are shown in red or blue, respectively, and the remaining Htx subjects are depicted in gray. (Upper) Probe intensities in consecutive 10-SNP windows normalized to a mean of 0 and SD of 1 from values in the remaining Htx subjects. P values supporting CNVs are shown in Table S1. Ratios of results of qPCR in index cases compared with controls are shown as red diamonds. (Lower) B-allele fraction (BAF) of SNPs across the interval. Arrows indicate locations of implicated LR genes. (A) Independent deletion and duplication affecting TGFBR2 in subjects 28 and 139, respectively. (B) Deletion of first three exons of GALNT11 in subject 257. (C) Duplication of ROCK2 in subject 152.
Fig. 3. WMISH analysis. (A–U) Results of in situ hybridization at three stages are shown for seven genes showing patterns of interest. These genes show expression in one or more of the following: GRP (blue arrows); heart or branchial arches (red arrows); kidney (green arrows). Stage 15 to 19 embryos are viewed dorso-posteriorly with anterior to the top to examine GRP expression (shown schematically in V). Stage 26 to 29 and stage 33 to 36 embryos are viewed laterally with anterior to the left (shown schematically in W and X, respectively).
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