Fakhro KA et al. (2011), Rare copy number variations in congenital heart...

XB-ART-42765

Proc Natl Acad Sci U S A. February 15, 2011; 108 (7): 2915-20.

Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning.

Fakhro KA , Choi M , Ware SM , Belmont JW , Towbin JA , Lifton RP , Khokha MK , Brueckner M .

Abstract
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 LR organizer (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.

PubMed ID: 21282601
PMC ID: PMC3041108
Article link: Proc Natl Acad Sci U S A.
Grant support: R01DE018824 NIDCR NIH HHS, R01DE18825 NIDCR NIH HHS, R01HD045789 NICHD NIH HHS , Howard Hughes Medical Institute

Genes referenced: aldh1a1 ccbl1 cryzl1 dnah9 galnt11 gdf7 greb1 grik1 ift88 igfbp5 laptm5 lrrc8a myh6 nek2 nup188 pdgfc pitx2 rho rock2 rpsa runx2 smarcal1 tgfbr2.2 tpk1 trat1
Antibodies referenced:
Morpholinos referenced: aldh1a1 MO1 ccbl1 MO1 cryzl1 MO1 dnah9 MO1 galnt11 MO1 gdf7 MO1 greb1 MO1 grik1 MO1 ift88 MO3 igfbp5 MO1 laptm5 MO1 lrrc8a MO1 myh6 MO1 nek2 MO1 nup188 MO1 pdgfc MO1 rock2 MO1 runx2 MO1 smarcal1 MO1 tgfbr2 MO1 tpk1 MO1 trat1 MO1

Article Images: [+] show captions

	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 left lateral plate mesoderm (LPM, arrow). (B) Same normal embryo showing absent pitx2 expression in the right LPM. (C and D) Absent pitx2 expression. No pitx2 mRNA is found in the left or right LPM. (E and F) Bilateral pitx2 expression. The pitx2 mRNA is found in both left and right LPM (arrows). (G and H) Right pitx2 expression: pitx2 mRNA is absent from the left LPM, present in the right LPM. (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) LPM pitx2 expression.
	ROCK2 (Rho-associated, coiled-coil containing protein kinase 2) gene expression in Xenopus laevis embryos, NF stage 33, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	lrrc8a (leucine rich repeat containing 8 family, member A) gene expression in Xenopus laevis embryos, NF stage 33, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	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: anterior left, dorsal up.

	NEK2(NIMA-related kinase 2) gene expression in Xenopus laevis embryos, NF stage 33, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	tgfbr2(transforming growth factor, beta receptor 2 (70/80kDa)) gene expression in Xenopus laevis embryos, NF stage 33, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.