Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.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 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).
Avidor-Reiss,
Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis.
2004,
Pubmed
Barber,
8p23.1 duplication syndrome; a novel genomic condition with unexpected complexity revealed by array CGH.
2008,
Pubmed
Bartoloni,
Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia.
2002,
Pubmed
Bisgrove,
Genetics of human laterality disorders: insights from vertebrate model systems.
2003,
Pubmed
Blum,
Xenopus, an ideal model system to study vertebrate left-right asymmetry.
2009,
Pubmed
,
Xenbase
Capelson,
Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes.
2010,
Pubmed
Choi,
Genetic diagnosis by whole exome capture and massively parallel DNA sequencing.
2009,
Pubmed
Cohen,
Controversies, genetics, diagnostic assessment, and outcomes relating to the heterotaxy syndrome.
2007,
Pubmed
Conrad,
Origins and functional impact of copy number variation in the human genome.
2010,
Pubmed
Craddock,
Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.
2010,
Pubmed
Dawe,
The Meckel-Gruber Syndrome proteins MKS1 and meckelin interact and are required for primary cilium formation.
2007,
Pubmed
De Luca,
Familial transposition of the great arteries caused by multiple mutations in laterality genes.
2010,
Pubmed
Eggenschwiler,
Cilia and developmental signaling.
2007,
Pubmed
Fry,
The Nek2 protein kinase: a novel regulator of centrosome structure.
2002,
Pubmed
,
Xenbase
Gebbia,
X-linked situs abnormalities result from mutations in ZIC3.
1997,
Pubmed
Greenway,
De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot.
2009,
Pubmed
Hellsten,
The genome of the Western clawed frog Xenopus tropicalis.
2010,
Pubmed
,
Xenbase
Herr,
Regulation of TGF-(beta) signalling by N-acetylgalactosaminyltransferase-like 1.
2008,
Pubmed
,
Xenbase
Iafrate,
Detection of large-scale variation in the human genome.
2004,
Pubmed
Jiao,
Tgfbeta signaling is required for atrioventricular cushion mesenchyme remodeling during in vivo cardiac development.
2006,
Pubmed
Kalverda,
Nucleoporins directly stimulate expression of developmental and cell-cycle genes inside the nucleoplasm.
2010,
Pubmed
Karkera,
Loss-of-function mutations in growth differentiation factor-1 (GDF1) are associated with congenital heart defects in humans.
2007,
Pubmed
Kennedy,
Congenital heart disease and other heterotaxic defects in a large cohort of patients with primary ciliary dyskinesia.
2007,
Pubmed
Khokha,
Techniques and probes for the study of Xenopus tropicalis development.
2002,
Pubmed
,
Xenbase
Kontaridis,
PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects.
2006,
Pubmed
Kosaki,
Characterization and mutation analysis of human LEFTY A and LEFTY B, homologues of murine genes implicated in left-right axis development.
1999,
Pubmed
Kosaki,
Left-right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB.
1999,
Pubmed
,
Xenbase
Laitenberger,
D-transposition of the great arteries in a case of microduplication 22q11.2.
2008,
Pubmed
Levin,
A molecular pathway determining left-right asymmetry in chick embryogenesis.
1995,
Pubmed
Loeys,
A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2.
2005,
Pubmed
Mahjoub,
NIMA-related kinases defective in murine models of polycystic kidney diseases localize to primary cilia and centrosomes.
2005,
Pubmed
McGrath,
Two populations of node monocilia initiate left-right asymmetry in the mouse.
2003,
Pubmed
Mefford,
Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes.
2008,
Pubmed
Mogi,
Xenopus neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein.
2003,
Pubmed
,
Xenbase
Nachury,
A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis.
2007,
Pubmed
Nonaka,
Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein.
1998,
Pubmed
Ostrowski,
A proteomic analysis of human cilia: identification of novel components.
2002,
Pubmed
Pazour,
Proteomic analysis of a eukaryotic cilium.
2005,
Pubmed
Piedra,
Pitx2 participates in the late phase of the pathway controlling left-right asymmetry.
1998,
Pubmed
Pierpont,
Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics.
2007,
Pubmed
Portnoï,
Microduplication 22q11.2: a new chromosomal syndrome.
2009,
Pubmed
Roessler,
Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly.
2009,
Pubmed
Schwabe,
Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations.
2008,
Pubmed
Schweickert,
Cilia-driven leftward flow determines laterality in Xenopus.
2007,
Pubmed
,
Xenbase
Schweickert,
The nodal inhibitor Coco is a critical target of leftward flow in Xenopus.
2010,
Pubmed
,
Xenbase
Shiratori,
The left-right axis in the mouse: from origin to morphology.
2006,
Pubmed
Sonn,
Suppression of Nek2A in mouse early embryos confirms its requirement for chromosome segregation.
2004,
Pubmed
Sutherland,
Disorders of left-right asymmetry: heterotaxy and situs inversus.
2009,
Pubmed
Taketazu,
Spectrum of cardiovascular disease, accuracy of diagnosis, and outcome in fetal heterotaxy syndrome.
2006,
Pubmed
Theerthagiri,
The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex.
2010,
Pubmed
,
Xenbase
Thienpont,
Duplications of the critical Rubinstein-Taybi deletion region on chromosome 16p13.3 cause a novel recognisable syndrome.
2010,
Pubmed
Tomita-Mitchell,
GATA4 sequence variants in patients with congenital heart disease.
2007,
Pubmed
van Bon,
Transposition of the great vessels in a patient with a 2.9 Mb interstitial deletion of 9q31.1 encompassing the inversin gene: clinical report and review.
2008,
Pubmed
Van der Aa,
Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome.
2009,
Pubmed
Vick,
Flow on the right side of the gastrocoel roof plate is dispensable for symmetry breakage in the frog Xenopus laevis.
2009,
Pubmed
,
Xenbase
Wang,
The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish.
2011,
Pubmed
Wei,
Rho kinases play an obligatory role in vertebrate embryonic organogenesis.
2001,
Pubmed
Zhang,
Rock2 controls TGFbeta signaling and inhibits mesoderm induction in zebrafish embryos.
2009,
Pubmed
