40 research outputs found
Autosomal recessive IFT57 hypomorphic mutation cause ciliary transport defect in unclassified oral-facial-digital syndrome with short stature and brachymesophalangia
No full textThe 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies
Comparison of <i>Ift140</i><sup><i>220</i></sup> allele and <i>Ift140</i><sup><i>null1</i></sup> allele.
No full text(A) Ift140 mRNA levels in MEFs. Levels of Ift140 transcript between exons 6 and 7 and between exons 13 and 14 was measured by qPCR. Wild type was set to 100%. For Ift140220/220, n = 1 control cell line and 2 mutant lines repeated 3 times. For Ift140null1/null1, n = 3 control lines and 3 mutant lines analyzed once each. *** p ≤ 0.001, Students t test. (B) Western blot analysis of IFT140 protein levels in MEFs from the 2 alleles. (C) MEFs from control and mutant lines stained for cilia (acetylated tubulin, red) and either IFT140 or IFT88 (green). 0% of Ift140null1/null1 cells showed IFT140 staining at the ciliary base or centrosome. 63 ± 16% of Ift140220/220 cells showed weak IFT140 staining at the ciliary base while control cells for each experiment showed 100% of the ciliated cells showing an IFT140 spot at the ciliary base. For Ift140220/220, n = 1 control cell line and 2 mutant lines repeated 3 times. For Ift140null1/null1, n = 3 control lines and 3 mutant lines analyzed once each. Scale bar is 5 microns. Arrows mark ciliary tip. (D) Percent ciliation in control and mutant fibroblast lines. For Ift140null1, n = 100 cells counted from 3 repeats of 1 control line and 1 mutant line. *****p t test. For Ift140220, n = 100 cells counted from 3 repeats of 1 control and 2 Ift140220/220 mutant lines. **p = 0.0037 t test. (E) Cilia on Bowman’s capsule of the kidney stained for centrosomes (γ-tubulin, red, arrow) and IFT88 (green). Scale bar is 5 microns. (F) Scanning EM images of control, Ift140220/220, and Ift140null1/null1 embryo nodes harvested at E7.5. Arrows indicate cilia. Scale bar is 5 microns. The data underlying this figure can be found in Supporting information S1 Data. MEF, mouse embryonic fibroblast.</p
3D reconstruction of a wild-type E16.5 embryo processed using episcopic confocal microscopy highlighting normal cardiac outflow tact development.
No full textAo: Aorta, dAo: Descending aorta, ASLV: Aortic semilunar valve, DA: Ductus arteriosus, LA: Left atria, LB: Left Bronchus, LPA: Left pulmonary artery, LV: Left ventricle, PA: Pulmonary artery, PSLV: Pulmonary semilunar valve, PT: Pulmonary trunk, RA: Right atria, RB: Right Bronchus, RCA: Right carotid artery, RPA: Right pulmonary artery, RV Right ventricle, T: Trachea. (MP4)</p
3D reconstruction of a wild-type E16.5 embryo processed using episcopic confocal microscopy highlighting normal Trachea/Esophagus development.
No full textAo: Aorta, dAo: Descending aorta, E: Esophagus, LA: Left atria, LB: Left Bronchus, LCA: Left carotid artery, LV: Left ventricle, MV: Mitral valve, PV: Pulmonary vein, RA: Right atria, RB: Right Bronchus, RCA: Right carotid artery, RV: Right ventricle, S: Stomach, SCV: Subclavian vein, T: Trachea, TV: Tricuspid valve, VC: Vena cava. (MP4)</p
Cardiac and great vessel defects with <i>Wnt1-Cre</i> or <i>Tbx18-Cre</i> deletion of <i>Ift140</i>.
No full text(A–F) Both Wnt1-Cre and Tbx18-Cre driven Ift140 deletion (Ift140flox/null1, Cre+) produced only mild congenital cardiac defects. All experimental hearts displayed normal heart looping and the majority had normal ventricular (A, D) and atrial (C, F) septum anatomy. However, a small number of Wnt1-Cre and Tbx18-Cre experimental animals displayed perimembranous ventricular septal defects (arrowheads in B, E). (G) Wnt1-Cre driven Ift140 deletion caused defects in great artery patterning including interrupted aorta (arrows in G), with or without the development of a long hypoplastic collateral vessel linking the left carotid artery and left subclavian artery (arrowheads in G). The development of anomalous right subclavian arteries was common in Wnt1-Cre experimental embryos (Ift140flox/null1, Wnt1-Cre). These arose from either the pulmonary trunk adjacent to the pulmonary arteries (§), as a vascular sling arising from the descending aorta and wrapping behind the trachea (‡), or as a vascular ring with attachments to both the pulmonary trunk and descending aorta (*). A: aorta; P: pulmonary trunk; LV: left ventricle; RV: right ventricle; LA: left atria; RA: right atria; T: trachea; RCA: right carotid artery; LCA: left carotid artery; LSA: left subclavian artery; RSA: right subclavian artery; dAo: descending aorta; RPA: right pulmonary artery; LPA: left pulmonary artery; T: trachea. All scales bars = 0.5 mm.</p
<i>Ift140</i><sup><i>null1/null1</i></sup> embryos display major anatomical defects at E9.5 and cardiac/great vessel patterning defects at E14.5.
No full text(A–I) 3D reconstructions of E9.5 Ift140+/+ control and littermate homozygous Ift140null1/null1 embryos analyzed by episcopic confocal microscopy. In A-B note the hypertrophy of the first branchial arch (1), and hypotrophy of the other branchial arches (2–4). In C-F note neural tube abnormalities characterized by the head fold failing to close in Ift140null1/null1 embryos (E-F). In G-I note the randomization of heart tube looping characterized by normal D-looped (G), reversed L-looped (H), and midline A-looped (I) heart tubes. (J–L) Sagittal section reconstructions of Ift140null1/null1 embryos further highlight the abnormal midline A-looped heart tube phenotype (L) observed in some Ift140null1/null1 embryos compared to embryos with D-looped (J) or L-looped (K) heart tubes. (M) Quantification of the Ift140null1/null1 heart looping defects reveals the randomization of looping phenotypes compared with wild-type embryos (n = 22). (N) Measurement of the OFT length in E10.5 embryos showed significant lengthening of the OFT in the Ift14O KO embryos. Data is mean ± SD. * p = 0.0007 assessed by unpaired Student t test. (O–V) Numerous cardiac and great vessel defects were seen in E14.5 Ift140null1/null1 embryos including: small ventricles (O vs. S), AVSDs with mutant embryos displaying a complete absence of normal atrial septum (arrow P vs. T), PTA characterized by a single OFT due to OFT failing to septate into separate aorta and pulmonary vessels (Q vs. U), and TEF characterized by a single unseptated tracheoesophageal tube (R vs. V). (W) Great vessel patterning was also perturbed in Ift140null1/null1 embryos (n = 7). Besides PTA, mutants showed a combination of singular or double left and right descending aortas. *: somites; 1, 2, 3, 4: Branchial arches; A: aorta; AVSD: atrioventricular septal defect; dA: descending aorta; f: forebrain; fl: forelimb; h: hindbrain; ht: heart tube; if: inflow tract; LA: left atrium; LCA: left carotid artery; LSA: left subclavian artery; LV: left ventricle; lv: liver; m: midbrain; o: esophagus; of: outflow tract; P: pulmonary trunk; PTA: persistent truncus arteriosus; RA: right atrium; RCA: right carotid artery; RV: right ventricle; t: trachea; tn: tongue; TEF: tracheoesophageal fistula; arrowhead: ventricular septal defect; arrow: atrial septum. Scales bars: A, B, J, K, L, N–U = 0.5 mm, C–I = 0.25 mm. The data underlying this figure can be found in supplemental file S1 Data.</p
<i>Ift140</i><sup><i>null1/null1</i></sup> embryos display major anatomical defects at E14.5.
No full textGross anatomical examination revealed numerous severe defects in E14.5 Ift140null1/null1 embryos (E–H) compared to controls (A–D) including significant hydrops (*), hypoplastic forelimbs (fl), hypoplastic maxillary region (mx), including reduced maxillary, medial and lateral nasal prominences resulting in bilateral cleft lip, hyperplastic mandibular region (md), missing abdominal walls and diaphragm with gastroschisis/ectopia cordis (F, G), smaller chests (D vs. H), and exencephaly with swollen neural tissue pouches surround an empty hollow cavity (‡). (I–L) 3D reconstitutions highlight the craniofacial defects (I, K) and polydactyly (J, L) found in E14.5 Ift140null1/null1 embryos. (M) Chest size was quantified by measuring chest areas that revealed that IFT140null1/null1 embryos (n = 7) displayed significantly smaller chests than age matched wild-type embryos (n = 3) (unpaired Students t test; p = 0.0065). cx: cerebral cortex; d: diaphragm; dA: descending aorta; e: eye; fb: forebrain; fl: forelimb; hl: hindlimb; hf: hair follicles; i: small intestine; ie: inner ear; ln: lung; lnp: lateral nasal prominences; lv: liver; mb: midbrain; md: mandibular region; mnp: medial nasal prominence; mx: maxillary region; ns: nasal capsule; op: otic placode; s: stomach; sc: spinal cord; t: trachea; tn: tongue; v: ventricle. Scale bars: A–I, K = 1 mm, J, L = 0.5 mm. The data underlying this figure can be found in Supporting information S1 Data.</p
Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes
No full textOral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype
Loss of IFT140 after tamoxifen treatment.
No full text(A) Western blot showing IFT140 levels in whole embryo lysates (Ift140flox/+ CAGGCre-ER+ vs. Ift140flox/null1 CAGGCre-ER+) 48 h after treatment of the mother with 0.1 ml (1 mg) tamoxifen administered by oral gavage. Embryos were treated at E9 and harvested at E11. γ-tubulin is a loading control. (B) Quantification of the extent of IFT140 reduction 48 h after treatment of the mother with tamoxifen. Level of IFT140 was normalized between embryos using γ-tubulin and then experimental and control embryos within a litter were ratioed with controls set to 100%. Raw counts were normalized to controls from the same litter. ***p t test. Error bar is standard deviation. The data underlying this figure can be found in Supporting information S1 Data. (TIF)</p
Late tamoxifen deletion of <i>Ift140</i> with <i>CAGGCre-ER</i> uncovers additional phenotypes.
No full textIft140flox/+, CAGGCre-ER+ (Control) (A, D, G, J, L) and Ift140flox/null1, CAGGCre-ER+ experimental (B, C, E, F, G, H, K, M, N) embryos were treated with tamoxifen at E7.5 or E8.5 and harvested at E16.5. E7.5 tamoxifen-dosed embryos show severe gastroschisis with the majority of the abdominal organs protruding from the abdominal cavity (A, B, D, E). E8.5 dosed embryos show only moderate gastroschisis with only some of the abdominal organs found outside of the abdominal cavity (A, C, D, F). Both E7.5 and E8.5 dosed embryos showed significant hydrops (* B, C, E, F), polydactyl (H, I), and hypoplastic lungs (B, C, E, F). Laterality defects were not observed in either E7.5 or E8.5 tamoxifen-dosed embryos, with all hearts displaying a normal D-looping phenotype (J, K). However, cardiac defects were observed in the experimental animals including ventricular septal defects (J, K) and AVSDs (L, M). (N) While the great vessels of both E7.5 and E8.5 tamoxifen-dosed experimental embryos displayed normally septated aorta and pulmonary trunk, approximately 50% had great artery patterning defects including: right aortic arch, interrupted aorta (arrows), hypoplastic transverse aorta (arrowhead), hypoplastic pulmonary arteries (*), and in 1 case double aortic arch with both left and right descending aortas. A: aorta; P: pulmonary trunk; LV: left ventricle; RV: right ventricle; LA: left atria; RA: right atria; t: trachea; o: esophagus; VSD: Ventricular septal defect; AVSD: atrioventricular septal defect; cx: cerebral cortex; sc: spinal cord; mb: midbrain; fl: forelimb; hl: hindlimb; e: eye; forebrain; op: otic placode; hf: hair follicles; RCA: right carotid artery; LCA: left carotid artery; LSA: left subclavian artery; LdAo: left descending aorta; RdAo: right descending aorta; RPA: right pulmonary artery; LPA: left pulmonary artery; Ao: aorta; P: pulmonary trunk; lv: liver; ln: lungs; s: stomach; i: small intestine. Scales bars: A–I = 1 mm, J–M = 0. 5 mm.</p
