Pedro A. Sanchez-Lara

NOTCH2 Mutations Cause Alagille Syndrome, a Heterogeneous Disorder of the Notch Signaling Pathway

Alagille syndrome (AGS) is caused by mutations in the gene for the Notch signaling pathway ligand Jagged1 (JAG1), which are found in 94% of patients. To identify the cause of disease in patients without JAG1 mutations, we screened 11 JAG1 mutation-negative probands with AGS for alterations in the gene for the Notch2 receptor (NOTCH2). We found NOTCH2 mutations segregating in two families and identified five affected individuals. Renal manifestations, a minor feature in AGS, were present in all the affected individuals. This demonstrates that AGS is a heterogeneous disorder and implicates NOTCH2 mutations in human disease.

Neuroimaging findings in macrocephaly–capillary malformation: A longitudinal study of 17 patients

Here, we report the neuroimaging findings and neurological changes in 17 unpublished patients with Macrocephaly–Capillary Malformation (M–CM). This syndrome has been traditionally known as Macrocephaly–Cutis Marmorata Telangiectatica Congenita (M–CMTC), but we explain why M–CM is a more accurate term for this overgrowth syndrome. We analyzed the 17 patients with available brain MRI or CT scans and compared their findings with features identified by a comprehensive review of published cases. White matter irregularities with increased signal on T2‐weighted images were commonly observed findings. A distinctive feature in more than half the patients was cerebellar tonsillar herniation associated with rapid brain growth and progressive crowding of the posterior fossa during infancy. In four such cases, we confirmed that the tonsillar herniation was an acquired event. Concurrently, with the development of these findings, ventriculomegaly (frequently obstructive) and dilated dural venous sinuses were observed in conjunction with prominent Virchow–Robin spaces in many of those in whom cerebellar tonsil herniation had developed. We postulate that this constellation of unusual features suggests a dynamic process of mechanical compromise in the posterior fossa, perhaps initiated by a rapidly growing cerebellum, which leads to congestion of the venous drainage with subsequently compromised cerebrospinal fluid reabsorption, all of which increases the posterior fossa pressure and leads to acquired tonsillar herniation. We make a distinction between congenital Chiari I malformation and acquired cerebellar tonsil herniation in this syndrome. We also observed numerous examples of abnormal cortical morphogenesis, including focal cortical dysplasia, polymicrogyria which primarily involved the perisylvian and insular regions, and cerebral and/or cerebellar asymmetric overgrowth. Other findings included a high frequency of cavum septum pellucidum or vergae, thickened corpus callosum, prominent optic nerve sheaths and a single case of venous sinus thrombosis. One patient was found to have a frontal perifalcine mass resembling a meningioma at age 5 years. This is the second apparent occurrence of this specific tumor in M–CM.

Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice

Patients with mutations in either TGF-β receptor type I (TGFBR1) or TGF-β receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-β signaling. Similarly, mutations in TGF-β receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-β ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-β2 and TGF-β receptor type III (TβRIII); activation of a TβRI/TβRIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted TβRI/TβRIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-β signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-β signaling may be beneficial for the prevention of congenital craniofacial birth defects.

Rhombencephalosynapsis: a hindbrain malformation associated with incomplete separation of midbrain and forebrain, hydrocephalus and a broad spectrum of severity

Rhombencephalosynapsis is a midline brain malformation characterized by missing cerebellar vermis with apparent fusion of the cerebellar hemispheres. Rhombencephalosynapsis can be seen in isolation or together with other central nervous system and extra-central nervous system malformations. Gómez-López-Hernández syndrome combines rhombencephalosynapsis with parietal/temporal alopecia and sometimes trigeminal anaesthesia, towering skull shape and dysmorphic features. Rhombencephalosynapsis can also be seen in patients with features of vertebral anomalies, anal atresia, cardiovascular anomalies, trachea-oesophageal fistula, renal anomalies, limb defects (VACTERL) association. Based on a comprehensive evaluation of neuroimaging findings in 42 patients with rhombencephalosynapsis, we propose a spectrum of severity, ranging from mild (the partial absence of nodulus, anterior and posterior vermis), to moderate (the absence of posterior vermis with some anterior vermis and nodulus present), to severe (the absence of posterior and anterior vermis with some nodulus present), to complete (the absence of the entire vermis including nodulus). We demonstrate that the severity of rhombencephalosynapsis correlates with fusion of the tonsils, as well as midbrain abnormalities including aqueductal stenosis and midline fusion of the tectum. Rhombencephalosynapsis is also associated with multiple forebrain abnormalities including absent olfactory bulbs, dysgenesis of the corpus callosum, absent septum pellucidum and, in rare patients, atypical forms of holoprosencephaly. The frequent association between rhombencephalosynapsis and aqueductal stenosis prompted us to evaluate brain magnetic resonance images in other patients with aqueductal stenosis at our institution, and remarkably, we identified rhombencephalosynapsis in 9%. Strikingly, subjects with more severe rhombencephalosynapsis have more severely abnormal neurodevelopmental outcome, as do subjects with holoprosencephaly and patients with VACTERL features. In summary, our data provide improved diagnostic and prognostic information, and support disruption of dorsal-ventral patterning as a mechanism underlying rhombencephalosynapsis.

A genome-wide association study identifies susceptibility loci for nonsyndromic sagittal craniosynostosis near BMP2 and within BBS9

Sagittal craniosynostosis is the most common form of craniosynostosis, affecting approximately one in 5,000 newborns. We conducted, to our knowledge, the first genome-wide association study for nonsyndromic sagittal craniosynostosis (sNSC) using 130 non-Hispanic case-parent trios of European ancestry (NHW). We found robust associations in a 120-kb region downstream of BMP2 flanked by rs1884302 (P = 1.13 × 10−14, odds ratio (OR) = 4.58) and rs6140226 (P = 3.40 × 10−11, OR = 0.24) and within a 167-kb region of BBS9 between rs10262453 (P = 1.61 × 10−10, OR = 0.19) and rs17724206 (P = 1.50 × 10−8, OR = 0.22). We replicated the associations to both loci (rs1884302, P = 4.39 × 10−31 and rs10262453, P = 3.50 × 10−14) in an independent NHW population of 172 unrelated probands with sNSC and 548 controls. Both BMP2 and BBS9 are genes with roles in skeletal development that warrant functional studies to further understand the etiology of sNSC.

Investigation of NRXN1 deletions: clinical and molecular characterization.

Deletions at 2p16.3 involving exons of NRXN1 are associated with susceptibility for autism and schizophrenia, and similar deletions have been identified in individuals with developmental delay and dysmorphic features. We have identified 34 probands with exonic NRXN1 deletions following referral for clinical microarray‐based comparative genomic hybridization. To more firmly establish the full phenotypic spectrum associated with exonic NRXN1 deletions, we report the clinical features of 27 individuals with NRXN1 deletions, who represent 23 of these 34 families. The frequency of exonic NRXN1 deletions among our postnatally diagnosed patients (0.11%) is significantly higher than the frequency among reported controls (0.02%; P = 6.08 × 10−7), supporting a role for these deletions in the development of abnormal phenotypes. Generally, most individuals with NRXN1 exonic deletions have developmental delay (particularly speech), abnormal behaviors, and mild dysmorphic features. In our cohort, autism spectrum disorders were diagnosed in 43% (10/23), and 16% (4/25) had epilepsy. The presence of NRXN1 deletions in normal parents and siblings suggests reduced penetrance and/or variable expressivity, which may be influenced by genetic, environmental, and/or stochastic factors. The pathogenicity of these deletions may also be affected by the location of the deletion within the gene. Counseling should appropriately represent this spectrum of possibilities when discussing recurrence risks or expectations for a child found to have a deletion in NRXN1.

Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice

Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFβ) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFβ signaling and IRF6 activity during palate formation. Here, we show that TGFβ signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFβ signaling mediator Smad4 (Smad4fl/fl;K14-Cre;Irf6+/R84C) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2fl/fl;K14-Cre mice, although the secondary palate of Irf6+/R84C and Smad4fl/fl;K14-Cre mice form normally. Furthermore, Smad4fl/fl;K14-Cre;Irf6+/R84C mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFβ/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2fl/fl;K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFβ-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice.

Transforming Growth Factor-β Regulates Basal Transcriptional Regulatory Machinery to Control Cell Proliferation and Differentiation in Cranial Neural Crest-derived Osteoprogenitor Cells

Transforming growth factor-β (Tgf-β) signaling is crucial for regulating craniofacial development. Loss of Tgf-β signaling results in defects in cranial neural crest cells (CNCC), but the mechanism by which Tgf-β signaling regulates bone formation in CNCC-derived osteogenic cells remains largely unknown. In this study, we discovered that Tgf-β regulates the basal transcriptional regulatory machinery to control intramembranous bone development. Specifically, basal transcription factor Taf4b is down-regulated in the CNCC-derived intramembranous bone in Tgfbr2fl/fl;Wnt1-Cre mice. Tgf-β specifically induces Taf4b expression. Moreover, small interfering RNA knockdown of Taf4b results in decreased cell proliferation and altered osteogenic differentiation in primary mouse embryonic maxillary mesenchymal cells, as seen in Tgfbr2 mutant cells. In addition, we show that Taf1 is decreased at the osteogenic initiation stage in the maxilla of Tgfbr2 mutant mice. Furthermore, small interfering RNA knockdown of Taf4b and Taf1 together in primary mouse embryonic maxillary mesenchymal cells results in up-regulated osteogenic initiator Runx2 expression, with decreased cell proliferation and altered osteogenic differentiation. Our results indicate a critical function of Tgf-β-mediated basal transcriptional factors in regulating osteogenic cell proliferation and differentiation in CNCC-derived osteoprogenitor cells during intramembranous bone formation.

Fibroblast Growth Factor 9 (FGF9)-Pituitary Homeobox 2 (PITX2) Pathway Mediates Transforming Growth Factor β (TGFβ) Signaling to Regulate Cell Proliferation in Palatal Mesenchyme during Mouse Palatogenesis

Cleft palate represents one of the most common congenital birth defects. Transforming growth factor β (TGFβ) signaling plays crucial functions in regulating craniofacial development, and loss of TGFβ receptor type II in cranial neural crest cells leads to craniofacial malformations, including cleft palate in mice (Tgfbr2fl/fl;Wnt1-Cre mice). Here we have identified candidate target genes of TGFβ signaling during palatal formation. These target genes were selected based on combining results from gene expression profiles of embryonic day 14.5 palates from Tgfbr2fl/fl;Wnt1-Cre mice and previously identified cleft palate phenotypes in genetically engineered mouse models. We found that fibroblast growth factor 9 (Fgf9) and transcription factor pituitary homeobox 2 (Pitx2) expressions are significantly down-regulated in the palate of Tgfbr2fl/fl;Wnt1-Cre mice, and Fgf9 and Pitx2 loss of function mutations result in cleft palate in mice. Pitx2 expression is down-regulated by siRNA knockdown of Fgf9, suggesting that Fgf9 is upstream of Pitx2. We detected decreased expression of both cyclins D1 and D3 in the palates of Tgfbr2fl/fl;Wnt1-Cre mice, consistent with the defect in cell proliferation. Significantly, exogenous FGF9 restores expression of cyclins D1 and D3 in a Pitx2-dependent manner and rescues the cell proliferation defect in the palatal mesenchyme of Tgfbr2fl/fl;Wnt1-Cre mice. Our study indicates that a TGFβ-FGF9-PITX2 signaling cascade regulates cranial neural crest cell proliferation during palate formation.

The morphogenesis of wormian bones: A study of craniosynostosis and purposeful cranial deformation†

Wormian bones are accessory bones that occur within cranial suture and fontanelles, most commonly within the posterior sutures. They occur more frequently in disorders that have reduced cranial ossification, hypotonia or decreased movement, thereby resulting in deformational brachycephaly. The frequency and location of wormian bones varies with the type and severity of cranial deformation practiced by ancient cultures. We considered the hypothesis that the pathogenesis of wormian bones may be due to environmental variations in dural strain within open sutures and fontanelles. In order to explore this further, we measured the cephalic index (CI) in 20 purposefully deformed pre‐Columbian skulls: 10 from Chichen Itza, Mexico, and 10 from Ancon, Peru, as well as 20 anatomically normal skulls used for medical school anatomy classes. We tested for a direct correlation between the CI and the number of wormian bones in skulls with varying degrees of brachycephalic cranial deformation and found no significant correlation. When the CI was grouped into three categories (normal (CI < 81), brachycephalic (CI 81‐93), and severely brachycephalic (CI > 93)) there was a trend toward increasing number of wormian bones as the skull became more brachycephalic (P = 0.039). A second part or our study tabulated the frequency and location of large wormian bones (greater than 1 cm) in 3‐dimentional computerized tomography (3D‐CT) scans from 207 cases of craniosynostosis and compared these data with published data on 485 normal dry skulls from a manuscript on wormian bones by Parker in 1905. Among cases of craniosynostosis, large wormian bones were significantly more frequent (117 out of 207 3D‐CT scans) than in dry skulls (131 out of 485). There was a 3.5 greater odds of developing a wormian bone with premature suture closure (P < 0.001). Midline synostosis, specifically metopic or sagittal synostosis, has more wormian bones in the midline, whereas unilateral lambdoidal or coronal synostosis more often had wormian bones on the contralateral side. Taken together, these data suggest that wormian bones may arise as a consequence of mechanical factors that spread sutures apart and affect dural strain within sutures and fontanelles.