Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment.

Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26-28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality.

Neuronal Gtf2i deletion alters mitochondrial and autophagic properties.

Gtf2i encodes the general transcription factor II-I (TFII-I), with peak expression during pre-natal and early post-natal brain development stages. Because these stages are critical for proper brain development, we studied at the single-cell level the consequences of Gtf2i's deletion from excitatory neurons, specifically on mitochondria. Here we show that Gtf2i's deletion resulted in abnormal morphology, disrupted mRNA related to mitochondrial fission and fusion, and altered autophagy/mitophagy protein expression. These changes align with elevated reactive oxygen species levels, illuminating Gtf2i's importance in neurons mitochondrial function. Similar mitochondrial issues were demonstrated by Gtf2i heterozygous model, mirroring the human condition in Williams syndrome (WS), and by hemizygous neuronal Gtf2i deletion model, indicating Gtf2i's dosage-sensitive role in mitochondrial regulation. Clinically relevant, we observed altered transcript levels related to mitochondria, hypoxia, and autophagy in frontal cortex tissue from WS individuals. Our study reveals mitochondrial and autophagy-related deficits shedding light on WS and other Gtf2i-related disorders.

¿Qué puede esconder el retraso global del desarrollo? A propósito de un caso de causa genética / What can Global Developmental Delay Conceal? Apropos of a Case with a Genetic Cause

Se presenta un niño de 6 años con antecedente de retraso del lenguaje que llevó a sus padres a realizar múltiples consultas. En un primer momento, su cuadro fue interpretado como parte de un retraso global del desarrollo. Posteriormente, el paciente presentó convulsiones y episodios de descompensación metabólica, comenzando desde entonces su seguimiento por los Servicios de neurología, genética y metabolismo. Finalmente, tras varios estudios complementarios, por medio de un exoma trío se arribó al diagnóstico de síndrome de microduplicación del cromosoma 7q11.23, lo que justifica tanto el retraso global de desarrollo del paciente como su clínica neurológica. (AU)

A six-year-old boy presents with a history of language delay that led his parents to make multiple consultations. At first, we interpreted his condition as part of a global developmental delay. Subsequently, the patient presented seizures and episodes of metabolic decompensation, and since then, he had to be followed up by neurology, genetics, and metabolism services. Finally, after several complementary studies, following a trio exome analysis, we diagnosed chromosome 7q11.23 microduplication syndrome, which explains his global developmental delay and neurological symptoms. (AU)

Extensive characterization of a Williams syndrome murine model shows Gtf2ird1-mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1, which encodes a transcription factor suggested to play a role in the behavioral profile of Williams syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A "complete deletion" mouse, heterozygously eliminating the syntenic Williams syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1, which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the complete deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams syndrome critical region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Oxytocin receptor activation does not mediate associative fear deficits in a Williams Syndrome model.

Williams Syndrome results in distinct behavioral phenotypes, which include learning deficits, anxiety, increased phobias and hypersociability. While the underlying mechanisms driving this subset of phenotypes is unknown, oxytocin (OT) dysregulation is hypothesized to be involved as some studies have shown elevated blood OT and altered OT receptor expression in patients. A "Complete Deletion" (CD) mouse, modeling the hemizygous deletion in Williams Syndrome, recapitulates many of the phenotypes present in humans. These CD mice also exhibit impaired fear responses in the conditioned fear task. Here, we address whether OT dysregulation is responsible for this impaired associative fear memory response. We show direct delivery of an OT receptor antagonist to the central nervous system did not rescue the attenuated contextual or cued fear memory responses in CD mice. Thus, increased OT signaling is not acutely responsible for this phenotype. We also evaluated OT receptor and serotonin transporter availability in regions related to fear learning, memory and sociability using autoradiography in wild type and CD mice. While no differences withstood correction, we identified regions that may warrant further investigation. There was a nonsignificant decrease in OT receptor expression in the lateral septal nucleus and nonsignificant lowered serotonin transporter availability in the striatum and orbitofrontal cortex. Together, these data suggest the fear conditioning anomalies in the Williams Syndrome mouse model are independent of any alterations in the oxytocinergic system caused by deletion of the Williams locus.

BAZ1B the Protean Protein.

The bromodomain adjacent to the zinc finger domain 1B (BAZ1B) or Williams syndrome transcription factor (WSTF) are just two of the names referring the same protein that is encoded by the WBSCR9 gene and is among the 26-28 genes that are lost from one copy of 7q11.23 in Williams syndrome (WS: OMIM 194050). Patients afflicted by this contiguous gene deletion disorder present with a range of symptoms including cardiovascular complications, developmental defects as well as a characteristic cognitive and behavioral profile. Studies in patients with atypical deletions and mouse models support BAZ1B hemizygosity as a contributing factor to some of the phenotypes. Focused analysis on BAZ1B has revealed this to be a versatile nuclear protein with a central role in chromatin remodeling through two distinct complexes as well as being involved in the replication and repair of DNA, transcriptional processes involving RNA Polymerases I, II, and III as well as possessing kinase activity. Here, we provide a comprehensive review to summarize the many aspects of BAZ1B function including its recent link to cancer.

Oxidative Stress in Down and Williams-Beuren Syndromes: An Overview.

Oxidative stress is the result of an imbalance in the redox state in a cell or a tissue. When the production of free radicals, which are physiologically essential for signaling, exceeds the antioxidant capability, pathological outcomes including oxidative damage to macromolecules, aberrant signaling, and inflammation can occur. Down syndrome (DS) and Williams-Beuren syndrome (WBS) are well-known and common genetic conditions with multi-systemic involvement. Their etiology is linked to oxidative stress with important causative genes, such as SOD-1 and NCF-1, respectively, of the diseases being primarily involved in the regulation of the redox state. Early aging, dementia, autoimmunity, and chronic inflammation are some of the main characteristics of these conditions that can be associated with oxidative stress. In recent decades, there has been a growing interest in the possible role of oxidative stress and inflammation in the pathology of these conditions. However, at present, few studies have investigated these correlations. We provide an overview of the current literature concerning the role of oxidative stress and oxidative damage in genetic syndromes with a focus on Down syndrome and WBS. We hope to provide new insights to improve the management of complications related to these diseases.

Differential Coassembly of α1-GABAARs Associated with Epileptic Encephalopathy.

GABAA receptors (GABAARs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren syndrome identified a frameshifting de novo variant in a major GABAAR gene, GABRA1 This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25 Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with ß3, but not with ß2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential ß subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition.SIGNIFICANCE STATEMENT GABAARs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain, and their dysfunction underlies many neurologic disorders, especially epilepsy. Here we report the characterization of an α1-GABAAR variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1 subunit cell surface expression with ß subunits resulting in severely reduced synaptic inhibition. Our study reveals that disease-inducing variants can affect GABAAR structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.

Everolimus Rescues the Phenotype of Elastin Insufficiency in Patient Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

OBJECTIVE: Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells-derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in rescuing the SMC phenotype in elastin insufficiency patients. Approach and Results: SMCs generated from induced pluripotent stem cells from 5 elastin insufficiency patients with severe recurrent vascular stenoses (3 Williams syndrome and 2 elastin mutations) were phenotypically immature, hyperproliferative, poorly responsive to endothelin, and exerted reduced tension in 3-dimensional smooth muscle biowires. Elastin mRNA and protein were reduced in SMCs from patients compared to healthy control SMCs. Fourteen drug candidates were tested on patient SMCs. Of the mammalian target of rapamycin inhibitors studied, everolimus restored differentiation, rescued proliferation, and improved endothelin-induced calcium flux in all patient SMCs except one Williams syndrome. Of the calcium channel blockers, verapamil increased SMC differentiation and reduced proliferation in Williams syndrome patient cells but not in elastin mutation patients and had no effect on endothelin response. Combination treatment with everolimus and verapamil was not superior to everolimus alone. Other drug candidates had limited efficacy. CONCLUSIONS: Everolimus caused the most consistent improvement in SMC differentiation, proliferation and in SMC function in patients with both syndromic and nonsyndromic elastin insufficiency, and offers the best candidate for drug repurposing for treatment of elastin insufficiency associated vasculopathy.

Stenosis coexists with compromised α1-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) is a rare disorder caused by a heterozygous deletion of 26-28 contiguous genes that affects the brain and cardiovascular system. Here, we investigated whether WBS affects aortic structure and function in the complete deletion (CD) mouse model harbouring the most common deletion found in WBS patients. Thoracic aortas from 3-4 months-old male CD mice and wild-type littermates were mounted in wire myographs or were processed for histomorphometrical analysis. Nitric oxide synthase (NOS) isoforms and oxidative stress levels were assessed. Ascending aortas from young adult CD mice showed moderate (50%) luminal stenosis, whereas endothelial function and oxidative stress were comparable to wild-type. CD mice showed greater contractions to KCl. However, α1-adrenergic contractions to phenylephrine, but not with a thromboxane analogue, were compromised. Decreased phenylephrine responses were not affected by selective inducible NOS blockade with 1400 W, but were prevented by the non-selective NOS inhibitor L-NAME and the selective neuronal NOS inhibitor SMTC. Consistently, CD mice showed increased neuronal NOS expression in aortas. Overall, aortic stenosis in CD mice coexists with excessive nNOS-derived NO signaling that compromises ascending aorta α1-adrenergic contractions. We suggest that increased neuronal NOS signaling may act as a physiological 'brake' against the detrimental effects of stenosis.

Mild macrocytosis in Williams-Beuren syndrome.

OBJECTIVE: To evaluate the occurrence and estimate the frequency of macrocytosis in Williams-Beuren syndrome (WBS). STUDY DESIGN: Complete blood count (CBC) data from 179 subjects with WBS aged 1-69 were collected, with common parameters assessed for trends. Z-transformed mean corpuscular volume (MCV) was compared with each laboratory's reference range as well as with control data from the National Health and Nutrition Examination Survey (NHANES) 2013-2014 data archives. RESULTS: Just over a third (35%) subjects had at least one recorded incidence of macrocytosis. In comparisons of CBC parameters with an expected population mean, MCV and MCH were greater than, while Hct and RDW were lower than, expected values. The distribution of erythrocyte MCV is shifted to the right in WBS compared to controls, as was the mean value. Despite this, anemia was absent, except in a single medically complex WBS subject. Though there was a paucity of data available of variables that could potentially cause an elevated MCV, no obvious etiology could be elucidated. CONCLUSIONS: Mild macrocytosis without anemia affects a moderate subset of WBS patients, leading to a rightward shift in the MCV distribution curve. Providers encountering isolated mild macrocytosis in WBS can consider observation over further workup.

Agenesis and Hypomyelination of Corpus Callosum in Mice Lacking Nsun5, an RNA Methyltransferase.

Williams-Beuren syndrome (WBS) is caused by microdeletions of 28 genes and is characterized by cognitive disorder and hypotrophic corpus callosum (CC). Nsun5 gene, which encodes cytosine-5 RNA methyltransferase, is located in the deletion loci of WBS. We have reported that single-gene knockout of Nsun5 (Nsun5-KO) in mice impairs spatial cognition. Herein, we report that postnatal day (PND) 60 Nsun5-KO mice showed the volumetric reduction of CC with a decline in the number of myelinated axons and loose myelin sheath. Nsun5 was highly expressed in callosal oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs) from PND7 to PND28. The numbers of OPCs and OLs in CC of PND7-28 Nsun5-KO mice were significantly reduced compared to wild-type littermates. Immunohistochemistry and Western blot analyses of myelin basic protein (MBP) showed the hypomyelination in the CC of PND28 Nsun5-KO mice. The Nsun5 deletion suppressed the proliferation of OPCs but did not affect transition of radial glial cells into OPCs or cell cycle exit of OPCs. The protein levels, rather than transcriptional levels, of CDK1, CDK2 and Cdc42 in the CC of PND7 and PND14 Nsun5-KO mice were reduced. These findings point to the involvement of Nsun5 deletion in agenesis of CC observed in WBS.

Bone involvement and mineral metabolism in Williams' syndrome.

CONTEXT: The previous studies suggested a possible increased risk of hypercalcaemia and reduced bone mineral density (BMD) in Williams' syndrome (WS). However, an extensive study regarding bone metabolism has never been performed. OBJECTIVE: To investigate bone health in young adults with WS. DESIGN: Cross-sectional study. SETTINGS: Endocrinology and Metabolic Diseases and Medical Genetic Units. PATIENTS: 29 WS young adults and 29 age- and sex-matched controls. MAIN OUTCOME MEASURES: In all subjects, calcium, phosphorus, bone alkaline phosphatase (bALP), parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHVitD), osteocalcin (OC), carboxyterminal cross-linking telopeptide of type I collagen (CTX), 24-h urinary calcium and phosphorus, femoral-neck (FN) and lumbar-spine (LS) BMD and vertebral fractures (VFx) were assessed. In 19 patients, serum fibroblast growth factor-23 (FGF23) levels were measured. RESULTS: WS patients showed lower phosphorus (3.1 ± 0.7 vs 3.8 ± 0.5 mg/dL, p = 0.0001) and TmP/GFR (0.81 ± 0.32 vs 1.06 ± 0.25 mmol/L, p = 0.001), and an increased prevalence (p = 0.005) of hypophosphoremia (34.5 vs 3.4%) and reduced TmP/GFR (37.9 vs 3.4%). Moreover, bALP (26.3 ± 8.5 vs 35.0 ± 8.0 U/L), PTH (24.5 ± 12.6 vs 33.7 ± 10.8 pg/mL), OC (19.4 ± 5.3 vs 24.5 ± 8.7 ng/mL), and FN-BMD (- 0.51 ± 0.32 vs 0.36 ± 0.32) were significantly lower (p < 0.05), while CTX significantly higher (401.2 ± 169.3 vs 322.3 ± 122.4 pg/mL, p < 0.05). Serum and urinary calcium and 25OHVitD levels, LS-BMD and VFx prevalence were comparable. No cases of hypercalcemia and suppressed FGF23 were documented. Patients with low vs normal phosphorus and low vs normal TmP/GFR showed comparable FGF23 levels. FGF23 did not correlate with phosphorus and TmP/GFR values. CONCLUSIONS: Adult WS patients have reduced TmP/GFR, inappropriately normal FGF23 levels and an uncoupled bone turnover with low femoral BMD.

Glucose and lipid metabolism, bone density, and body composition in individuals with Williams syndrome.

OBJECTIVE: We assessed body composition, bone mineral density (BMD), glucose and lipids in Williams syndrome (WS), a rare microdeletion disorder. DESIGN: Individuals with WS had outpatient assessment at Massachusetts General Hospital. Controls were selected from the National Health and Nutrition Examination Survey (NHANES 2005-2006). PATIENTS: A total of 22 individuals with WS, each matched by age, sex and race to four NHANES controls. MEASUREMENTS: Blood sampling, oral glucose tolerance test, dual-energy X-ray absorptiometry scan. RESULTS: WS and control groups were 59% female and 29 ± 8 years old. Compared to controls, individuals with WS were shorter but had similar body weight, with more fat and less lean mass. Per cent body fat was higher in WS even after adjusting for BMI (+2.1% [95% CI 0.4, 3.9%]). Four WS patients had abnormal lower extremity fat accumulation resembling lipedema. HbA1c (+0.5% [0.2, 0.7]) and 2-hour glucose (+68 mg/dL [44, 93]) were higher in WS vs controls, differences which persisted after adjusting for BMI. Fasting glucose was comparable between groups. LDL (-18 mg/dL [-35, -2]) and triglycerides (-45 mg/dL [-87, -2]) were significantly lower in WS. Whole-body BMD was significantly lower (-0.15 g/cm2 [-0.20, -0.11]) in WS, and this remained true controlling for height (-0.06 g/cm2 [-0.11, -0.02]). Vitamin D was <30 ng/mL in 81% of those with WS. CONCLUSIONS: On average, adults with WS have increased fat, decreased lean mass, impaired glucose homeostasis and reduced BMD. Clinical efforts to build muscle and bone mass, and to ensure vitamin D sufficiency, are warranted. Genotype-phenotype research efforts are also warranted.

NMR assignments of the WBSCR27 protein related to Williams-Beuren syndrome.

Williams-Beuren syndrome is a genetic disorder characterized by physiological and mental abnormalities, and is caused by hemizygous deletion of several genes in chromosome 7. One of the removed genes encodes the WBSCR27 protein. Bioinformatic analysis of the sequence of WBSCR27 indicates that it belongs to the family of SAM-dependent methyltransferases. However, exact cellular functions of this protein or phenotypic consequences of its deficiency are still unknown. Here we report nearly complete 1H, 15N, and 13C chemical shifts assignments of the 26 kDa WBSCR27 protein from Mus musculus in complex with the cofactor S-adenosyl-L-methionine (SAM). Analysis of the assigned chemical shifts allowed us to characterize the protein's secondary structure and backbone dynamics. The topology of the protein's fold confirms the assumption that the WBSCR27 protein belongs to the family of class I methyltransferases.

Epigallocatechin-3-gallate improves cardiac hypertrophy and short-term memory deficits in a Williams-Beuren syndrome mouse model.

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a heterozygous deletion of 26-28 genes at chromosome band 7q11.23. The complete deletion (CD) mouse model mimics the most common deletion found in WBS patients and recapitulates most neurologic features of the disorder along with some cardiovascular manifestations leading to significant cardiac hypertrophy with increased cardiomyocytes' size. Epigallocatechin-3-gallate (EGCG), the most abundant catechin found in green tea, has been associated with potential health benefits, both on cognition and cardiovascular phenotypes, through several mechanisms. We aimed to investigate the effects of green tea extracts on WBS-related phenotypes through a phase I clinical trial in mice. After feeding CD animals with green tea extracts dissolved in the drinking water, starting at three different time periods (prenatal, youth and adulthood), a set of behavioral tests and several anatomical, histological and molecular analyses were performed. Treatment resulted to be effective in the reduction of cardiac hypertrophy and was also able to ameliorate short-term memory deficits of CD mice. Taken together, these results suggest that EGCG might have a therapeutic and/or preventive role in the management of WBS.

Impaired glucose metabolism in subjects with the Williams-Beuren syndrome: A five-year follow-up cohort study.

OBJECTIVE: The Williams-Beuren syndrome (WS) is associated with impaired glucose metabolism (IGM) early in adulthood. However, the pathophysiology of IGM remains poorly defined, due to the lack of longitudinal studies investigating the contribution of ß-cell dysfunction and impaired insulin sensitivity. This study aimed at assessing incidence of IGM and the underlying mechanisms in WS adults. METHODS: This observational, longitudinal (5-year), cohort study enrolled thirty-one consecutive WS subjects attending a tertiary referral center. An oral glucose tolerance test (OGTT) was performed yearly and used to classify patients as normal or IGM, including impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) and diabetes mellitus (DM), and to calculate surrogate measures of insulin secretion and/or sensitivity. RESULTS: IGM patients were 18 (58.1%, three DM) at baseline and 19 (61.3%, five DM) at end-of-follow-up. However, 13 individuals changed category of glucose homeostasis in both directions during follow-up (8 progressors, 5 regressors) and 18 did not (8 non-progressors, 10 non-regressors). New cases of IGM and DM were 11.1 and 2.53 per 100 persons-year, respectively, and were treated non-pharmacologically. In the whole cohort and, to a higher extent, in progressors, indices of early-phase insulin secretion and insulin sensitivity decreased significantly from baseline to end-of-follow-up, with concurrent reduction of the oral disposition index and insulin secretion-sensitivity index-2 (ISSI-2), compensating insulin secretion for the level of insulin resistance. No baseline measure independently predicted progression, which correlated with change from baseline in ISSI-2. Compared with patients with normal glucose homeostasis, IGT subjects had impaired insulin sensitivity, whereas insulin secretion was reduced only in those with IFG+IGT or DM. CONCLUSIONS: IGM incidence is high in young adults with WS, suggesting the need of early screening and timed intervention. As in classical type 2 diabetes, impaired insulin sensitivity and ß-cell dysfunction contribute, in this sequence, to progression to IGM and DM.

Deficient Circumferential Growth Is the Primary Determinant of Aortic Obstruction Attributable to Partial Elastin Deficiency.

OBJECTIVE: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN, the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined. APPROACH AND RESULTS: We quantified determinants of luminal stenosis in thoracic aortas of Eln-/- mice incompletely rescued by human ELN. Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject. CONCLUSIONS: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency.

DNA damage response defect in Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS, no. OMIM 194050) is a rare multisystem genetic disorder caused by a microdeletion on chromosome 7q11.23 and characterized by cardiovascular malformations, mental retardation, and a specific facial dysmorphism. Recently, we reported that a series of non­Hodgkin's lymphoma occurs in children with WBS and thus hypothesized that a predisposition to cancer may be associated with this genetic disorder. The aim of the present study was to ascertain the role played by three genes hemizygously deleted in WBS (RFC2, GTF2I and BAZ1B) in DNA damage response pathways. Cell proliferation, cell cycle analysis, γ­H2A.X induction, and expression of DNA damage response proteins were investigated upon exposure to genotoxic treatments in WBS patient­derived primary fibroblasts and in the 293T cell line treated with specific siRNAs targeting RFC2, GTF2I and BAZ1B. An impaired hydroxyurea­induced phosphorylation of CHK1 was observed in the WBS cells. However, this defective DNA damage response was not associated with an increased sensitivity to genotoxic agents. In addition, depletion of RFC2, GTF2I and BAZ1B using specific siRNAs did not have a significant impact on the DNA damage response in 293T cells. Our results highlight that the ATR­dependent DNA damage response is impaired in WBS patient cells but is also dispensable for viability when these cells undergo a genotoxic stress. The mechanism by which the ATR pathway is impaired in WBS warrants elucidation through further investigation.

RNA-Seq analysis of Gtf2ird1 knockout epidermal tissue provides potential insights into molecular mechanisms underpinning Williams-Beuren syndrome.

BACKGROUND: Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology. RESULTS: Using a targeted Gtf2ird1 knockout mouse, we employed massively-parallel sequencing of mRNA (RNA-Seq) to understand changes in the transcriptional landscape associated with inactivation of Gtf2ird1 in lip tissue. We found widespread dysregulation of genes including differential expression of 78 transcription factors or coactivators, several involved in organ development including Hey1, Myf6, Myog, Dlx2, Gli1, Gli2, Lhx2, Pou3f3, Sox2, Foxp3. We also found that the absence of GTF2IRD1 is associated with increased expression of genes involved in cellular proliferation, including growth factors consistent with the observed phenotype of extreme thickening of the epidermis. At the same time, there was a decrease in the expression of genes involved in other signalling mechanisms, including the Wnt pathway, indicating dysregulation in the complex networks necessary for epidermal differentiation and facial skin patterning. Several of the differentially expressed genes have known roles in both tissue development and neurological function, such as the transcription factor Lhx2 which regulates several genes involved in both skin and brain development. CONCLUSIONS: Gtf2ird1 inactivation results in widespread gene dysregulation, some of which may be due to the secondary consequences of gene regulatory network disruptions involving several transcription factors and signalling molecules. Genes involved in growth factor signalling and cell cycle progression were identified as particularly important for explaining the skin dysmorphology observed in this mouse model. We have noted that a number of the dysregulated genes have known roles in brain development as well as epidermal differentiation and maintenance. Therefore, this study provides clues as to the underlying mechanisms that may be involved in the broader profile of WBS.