Timothy C. Cox

MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders

BackgroundPatients with Opitz GBBB syndrome present with a variable array of developmental defects including craniofacial, cardiac, and genital anomalies. Mutations in the X-linked MID1 gene, which encodes a microtubule-binding protein, have been found in ~50% of Opitz GBBB syndrome patients consistent with the genetically heterogeneous nature of the disorder. A protein highly related to MID1, called MID2, has also been described that similarly associates with microtubules.ResultsTo identify protein partners of MID1 and MID2 we undertook two separate yeast two-hybrid screens. Using this system we identified Alpha 4, a regulatory subunit of PP2-type phosphatases and a key component of the rapamycin-sensitive signaling pathway, as a strong interactor of both proteins. Analysis of domain-specific deletions has shown that the B-boxes of both MID1 and MID2 mediate the interaction with Alpha 4, the first demonstration in an RBCC protein of a specific role for the B-box region. In addition, we show that the MID1/2 coiled-coil motifs mediate both homo- and hetero-dimerisation, and that dimerisation is a prerequisite for association of the MID-Alpha 4 complex with microtubules.ConclusionsOur findings not only implicate Alpha 4 in the pathogenesis of Opitz GBBB syndrome but also support our earlier hypothesis that MID2 is a modifier of the X-linked phenotype. Of further note is the observation that Alpha 4 maps to Xq13 within the region showing linkage to FG (Opitz-Kaveggia) syndrome. Overlap in the clinical features of FG and Opitz GBBB syndromes warrants investigation of Alpha 4 as a candidate for causing FG syndrome.

Heterozygous mutations of FREM1 are associated with an increased risk of isolated metopic craniosynostosis in humans and mice

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia.

Manitoba-oculo-tricho-anal (MOTA) syndrome is caused by mutations in FREM1

Background Manitoba-oculo-tricho-anal (MOTA) syndrome is a rare condition defined by eyelid colobomas, cryptophthalmos and anophthalmia/microphthalmia, an aberrant hairline, a bifid or broad nasal tip, and gastrointestinal anomalies such as omphalocele and anal stenosis. Autosomal recessive inheritance had been assumed because of consanguinity in the Oji-Cre population of Manitoba and reports of affected siblings, but no locus or cytogenetic aberration had previously been described. Methods and results This study shows that MOTA syndrome is caused by mutations in FREM1, a gene previously mutated in bifid nose, renal agenesis, and anorectal malformations (BNAR) syndrome. MOTA syndrome and BNAR syndrome can therefore be considered as part of a phenotypic spectrum that is similar to, but distinct from and less severe than, Fraser syndrome. Re-examination of Frem1bat/bat mutant mice found new evidence that Frem1 is involved in anal and craniofacial development, with anal prolapse, eyelid colobomas, telecanthus, a shortened snout and reduced philtral height present in the mutant mice, similar to the human phenotype in MOTA syndrome. Conclusions The milder phenotypes associated with FREM1 deficiency in humans (MOTA syndrome and BNAR syndrome) compared to that resulting from FRAS1 and FREM2 loss of function (Fraser syndrome) are also consistent with the less severe phenotypes resulting from Frem1 loss of function in mice. Together, Fraser, BNAR and MOTA syndromes constitute a clinically overlapping group of FRAS–FREM complex diseases.

Expression profiles of cIRF6, cLHX6 and cLHX7 in the facial primordia suggest specific roles during primary palatogenesis

BackgroundThe LIM-homeodomain transcription factors LHX7 and LHX6 have been implicated in palatogenesis in mice and thus may also contribute to the incidence of isolated palatal clefts and/or clefts of the lip and primary palate (CL/P) in humans. Causative mutations in the transcription factor IRF6 have also been identified in two allelic CL/P syndromes and common polymorphisms in the same gene are significantly associated with non-syndromal CL/P in different populations.ResultsHere we report the isolation of chick orthologues of LHX7, LHX6 and IRF6 and the first characterisation of their profiles of expression during morphogenesis of the midface with emphasis on the period around formation of the primary palate. LHX7 and LHX6 expression was restricted to the ectomesenchyme immediately underlying the ectoderm of the maxillary and mandibular primordia as well as to the lateral globular projections of the medial nasal process, again underlying the pre-fusion primary palatal epithelia. In contrast, IRF6 expression was restricted to surface epithelia, with elevated levels around the frontonasal process, the maxillary primordia, and the nasal pits. Elsewhere, high expression was also evident in the egg tooth primordium and in the apical ectodermal ridge of the developing limbs.ConclusionThe restricted expression of both LHX genes and IRF6 in the facial primordia suggests roles for these gene products in promoting directed outgrowth and fusion of the primary palate. The manipulability, minimal cost and susceptibility of chicks to CL/P will enable more detailed investigations into how perturbations of IRF6, LHX6 and LHX7 contribute to common orofacial clefts.

Comparing Canal Transportation and Centering Ability of EndoSequence and Vortex Rotary Files by Using Micro–Computed Tomography

INTRODUCTIONnEndoSequence and Vortex are 2 recently developed rotary file systems that are made with traditional nickel-titanium (NiTi) and M-Wire technology, respectively. Previous studies have demonstrated better fatigue resistance of M-Wire rotary files compared with the conventional NiTi file. However, no study has compared the effects of M-Wire and conventional NiTi on transportation and centering ability. Hence, the aim of this study was to evaluate the transportation and centering ability of EndoSequence and Vortex files in mesial roots of mandibular molars by using micro-computed tomography imaging.nnnMETHODSnSixteen extracted mandibular molars with mesiobuccal and mesiolingual canals with separate foramina were used. Preinstrumentation scans of all teeth were taken, and the teeth were divided into 2 groups. In group 1, the mesiobuccal canals were instrumented with Vortex files and the mesiolingual canals with EndoSequence files. In group 2, the mesiobuccal canals were instrumented with EndoSequence files and the mesiolingual canals with Vortex files. Two file sizes were compared, 30/.04 and 40/.04. Postinstrumentation scans were performed, and the 2 scans were compared to determine centering ability and transportation.nnnRESULTSnThe amount of transportation at 1, 3, and 5 mm was similar for both file types in both file sizes. Transportation toward the furcation area at 7 mm was greater with the 30/.04 Endosequence files compared with the Vortex 30/.04 files (P < .05), but there was no difference in size 40/.04 files.nnnCONCLUSIONSnOverall, our study does not support the use of one rotary file system over the other (Vortex or EndoSequence) when comparing transportation and centering ability.

Preferential Associated Anomalies in 818 Cases of Microtia in South America

The etiology of microtia remains unknown in most cases. The identification of patterns of associated anomalies (i.e., other anomalies that occur with a given congenital anomaly in a higher than expected frequency), is a methodology that has been used for research into the etiology of birth defects. We conducted a study based on cases of microtia that were diagnosed from more than 5 million live (LB)‐ and stillbirths (SB) examined in hospitals participating in ECLAMC (Latin American Collaborative Study of Congenital Malformations) between 1967 and 2009. We identified 818 LB and SB with microtia and at least one additional non‐related major congenital anomaly (cases) and 15,969 LB and SB with two or more unrelated major congenital anomalies except microtia (controls). A logistic regression analysis was performed to identify the congenital anomalies preferentially associated with microtia. Preferential associations were observed for 10 congenital anomalies, most of them in the craniofacial region, including facial asymmetry, choanal atresia, and eyelid colobomata. The analysis by type of microtia showed that for anomalies such as cleft lip and palate, macrostomia, and limb reduction defects, the frequency increased with the severity of the microtia. In contrast, for other anomalies the frequency tended to be the same across all types of microtia. Based on these results we will integrate data on the developmental pathways related to preferentially associated congenital anomalies for future studies investigating the etiology of microtia.

A distal 594 bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by the Hox-Pbx-Meis complex

Hmx1 encodes a homeodomain transcription factor expressed in the developing lateral craniofacial mesenchyme, retina and sensory ganglia. Mutation or mis-regulation of Hmx1 underlies malformations of the eye and external ear in multiple species. Deletion or insertional duplication of an evolutionarily conserved region (ECR) downstream of Hmx1 has recently been described in rat and cow, respectively. Here, we demonstrate that the impact of Hmx1 loss is greater than previously appreciated, with a variety of lateral cranioskeletal defects, auriculofacial nerve deficits, and duplication of the caudal region of the external ear. Using a transgenic approach, we demonstrate that a 594u2005bp sequence encompassing the ECR recapitulates specific aspects of the endogenous Hmx1 lateral facial expression pattern. Moreover, we show that Hoxa2, Meis and Pbx proteins act cooperatively on the ECR, via a core 32u2005bp sequence, to regulate Hmx1 expression. These studies highlight the conserved role for Hmx1 in BA2-derived tissues and provide an entry point for improved understanding of the causes of the frequent lateral facial birth defects in humans. Summary: The transcription factors Hoxa2, Meis and Pbx act cooperatively on an evolutionarily conserved region downstream of Hmx1 to regulate Hmx1 expression and craniofacial development.

Pulmonary Function Assessment in an Infant With Barnes Syndrome: Proactive Evaluation for Surgical Intervention

Our aim for this study was to report pulmonary mechanics in a neonate with a severe case of Barnes syndrome, a rare form of thoracolaryngopelvic dysplasia, and to use these data to guide ventilatory support and serve as a presurgical screening tool. A comprehensive pulmonary function evaluation was performed on a 36-day-old patient with Barnes syndrome who was being mechanically ventilated because of severe pulmonary distress secondary to thoracic dystrophy. The measurements consisted of respiratory volumes including functional residual capacity, ventilatory mechanics including compliance and resistance, and thoracoabdominal synchrony. Chest wall compliance was 64% below normal, and the thoracoabdominal motion was indicative of predominantly abdominal displacement during inspiratory breaths. The lungs were functioning at a low functional residual capacity, resulting in low lung compliance and increased pulmonary resistance. As a result of the evaluation, the patient was recommended for lateral thoracic expansion surgery and the ventilatory management was adjusted to focus on end-distending pressure support.

Microtomographic analysis of lower urinary tract obstruction.

Prenatal obstruction of the lower urinary tract may result in megacystis, with subsequent development of hydroureter, hydronephrosis, and renal damage. Oligo- or anhydramnios, pulmonary hypoplasia, and prune belly syndrome are lethal consequences. Causes and mechanisms responsible for obstruction remain unclear but might be clarified by anatomic study at autopsy. To this end, we employed 2 methods of tomographic imaging—optical projection tomography and contrast-enhanced microCT scanning—to elucidate the anatomy of the intact urinary bladder and urethra in 10 male fetuses with lower urinary tract obstruction. Images were compared with those from 9 age-matched controls. Three-dimensional images, rotated and sectioned digitally in multiple planes, permitted thorough examination while preserving specimens for later study. Both external and internal features of the bladder and urethra were demonstrated; small structures (ie, urethral crest, verumontanum, prostatic utricle, ejaculatory ducts) were seen in detail. Types of obstruction consisted of urethral atresia (n = 5), severe urethral stenosis (n = 2), urethral diaphragm (n = 2), or physical kinking (n = 1); classic (Young type I) posterior urethral valves were not encountered. Traditional light microscopy was then used to verify tomographic findings. The prostate gland was hypoplastic or absent in all cases; in 1, prostatic tissue was displaced inferior to the verumontanum. Findings support previous views that dissection may produce valve-like artifacts (eg, bisection of an obstructing diaphragm) and that deformation of an otherwise normal urethra may result in megacystis. The designation “posterior urethral valves” should not be used as a generic expression of urethral obstruction unless actual valves are demonstrated.

A translational cellular model to study the impact of high-frequency oscillatory ventilation on human epithelial cell function

High-frequency oscillatory ventilation (HFOV) has been proposed as gentle ventilation strategy to prevent lung injury in the preterm infant. High-frequency jet ventilation leads to dimensional and mechanical airway deformation in animal airway models, which is consistent with translational studies demonstrating the impact of oxygen and biophysical stresses on normal airway cellular function. There is an overall paucity of clinical and cellular data on the impact of HFOV on the conducting airway. We developed an innovative method to test the impact of the clinical HFO Ventilator (SensorMedics 3100A) on human epithelial cell function. In this translational model, we were able to study the differential effects of biophysical stress due to HFOV independently and in combination with hyperoxia on a direct cellular level of the conducting airway system. Additionally, we could demonstrate that hyperoxia and pressure by HFOV independently resulted in significant cell dysfunction and inflammation, while the combination of HFOV and hyperoxia had a synergistic effect, resulting in greater cell death.nnnNEW & NOTEWORTHYnTraditionally, large-animal models are used to analyze the impact of clinical ventilators on lung cellular function. In our dual-chamber model, we interface high-frequency oscillatory ventilation (HFOV) directly with airway cells to study the effects of HFOV independently and combined with hyperoxia. Therefore, it is possible to study the preclinical impact of interventional factors without the high cost of animal models, thus reducing staff, time, as well as animal sparing.