Novel SYNGAP1 Variant in an Adult Individual Affected by Intellectual Disability and Epilepsy: A Cold Case Solved through Whole-Exome Sequencing.

Introduction: Nowadays, whole-exome sequencing (WES) analysis is an essential part in the diagnostic pathway of individuals with complex phenotypes when routine exams, such as array-CGH and gene panels, have proved inconclusive. However, data on the diagnostic rate of WES analysis in adult individuals, negative to first-tier tests, are lacking. This is because initiatives with the aim of diagnosing rare diseases focus mainly on pediatric unsolved cases. Case Presentation: We hereby present a 45-year-old woman with severe intellectual disability, previous psychomotor developmental delay, behavioral disorders, stereotypies, nonconvulsive epilepsy, and dysmorphisms. The proband first came to our attention when she was 4 years old (in 1982); since then, she has undergone several clinical and instrumental assessments, without reaching a genetic diagnosis. At last, through WES analysis, a novel de novo variant in SYNGAP1 was found. The clinical characteristics associated with SYNGAP1 are similar to those presented by the proband. Conclusion: The variant is predicted to be deleterious and is most probably the cause of the proband's phenotype. The perseverance of the clinicians and the family allowed us to reach a diagnosis in a woman with a more than 30-year history of clinical evaluations, instrumental assessments, and genetic tests. This diagnosis was of significant relevance in genetic counseling for family members and the proband herself.

Genetic Downregulation of the Metabotropic Glutamate Receptor Type 5 Dampens the Reactive and Neurotoxic Phenotype of Adult ALS Astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100ß and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.

An example of parenchymal renal sparing in the context of complex malformations due to a novel mutation in the PBX1 gene.

INTRODUCTION: PBX1 encodes the pre-B cell leukemia factor 1, a Three Amino acid Loop Extension (TALE) transcription factor crucial to regulate basic developmental processes. PBX1 loss-of-function variants have been initially described in association with renal malformations in both isolated and syndromic forms. CASE REPORT: Herein, we report a male infant presenting multiple organ malformations (cleidosternal dysostosis, micrognathia, left lung hypoplasia, wide interatrial defect, pulmonary hypertension, total anomalous pulmonary venous return, intestinal malrotation) and carrying the heterozygous de novo c.868C > T (p.Arg290Trp) variant in PBX1. This novel variant affects the highly conserved homeodomain of the protein, leading to a non-conservative substitution and consequently altering its tridimensional structure and DNA-binding capacity. CONCLUSION: So far, PBX1 has been reported in association with a broad spectrum of renal anomalies. However, given the role of this gene in many different developing processes, whole-exome sequencing can detect mutations in PBX1 even in patients with different phenotypes, not necessarily involving the renal primordium. This report presents a novel PBX1 variant with a predicted strong deleterious effect. The mutation leads to a non-conservative substitution in a very highly conserved domain of the protein, thus altering its tertiary structure and DNA-binding capacity.

Neurodevelopmental Disorders in Patients With Complex Phenotypes and Potential Complex Genetic Basis Involving Non-Coding Genes, and Double CNVs.

Neurodevelopmental disorders (NDDs) are a heterogeneous class of brain diseases, with a complex genetic basis estimated to account for up to 50% of cases. Nevertheless, genetic diagnostic yield is about 20%. Array-comparative genomic hybridization (array-CGH) is an established first-level diagnostic test able to detect pathogenic copy number variants (CNVs), however, most identified variants remain of uncertain significance (VUS). Failure of interpretation of VUSs may depend on various factors, including complexity of clinical phenotypes and inconsistency of genotype-phenotype correlations. Indeed, although most NDD-associated CNVs are de novo, transmission from unaffected parents to affected children of CNVs with high risk for NDDs has been observed. Moreover, variability of genetic components overlapped by CNVs, such as long non-coding genes, genomic regions with long-range effects, and additive effects of multiple CNVs can make CNV interpretation challenging. We report on 12 patients with complex phenotypes possibly explained by complex genetic mechanisms, including involvement of antisense genes and boundaries of topologically associating domains. Eight among the 12 patients carried two CNVs, either de novo or inherited, respectively, by each of their healthy parents, that could additively contribute to the patients' phenotype. CNVs overlapped either known NDD-associated or novel candidate genes (PTPRD, BUD13, GLRA3, MIR4465, ABHD4, and WSCD2). Bioinformatic enrichment analyses showed that genes overlapped by the co-occurring CNVs have synergistic roles in biological processes fundamental in neurodevelopment. Double CNVs could concur in producing deleterious effects, according to a two-hit model, thus explaining the patients' phenotypes and the incomplete penetrance, and variable expressivity, associated with the single variants. Overall, our findings could contribute to the knowledge on clinical and genetic diagnosis of complex forms of NDD.

Case Report: Whole Exome Sequencing Revealed Disease-Causing Variants in Two Genes in a Patient With Autism Spectrum Disorder, Intellectual Disability, Hyperactivity, Sleep and Gastrointestinal Disturbances.

Autism Spectrum Disorder (ASD) refers to a broad range of conditions characterized by difficulties in communication, social interaction and behavior, and may be accompanied by other medical or psychiatric conditions. Patients with ASD and comorbidities are often difficult to diagnose because of the tendency to consider the multiple symptoms as the presentation of a complicated syndromic form. This view influences variant filtering which might ignore causative variants for specific clinical features shown by the patient. Here we report on a male child diagnosed with ASD, showing cognitive and motor impairments, stereotypies, hyperactivity, sleep, and gastrointestinal disturbances. The analysis of whole exome sequencing (WES) data with bioinformatic tools for oligogenic diseases helped us to identify two major previously unreported pathogenetic variants: a maternally inherited missense variant (p.R4122H) in HUWE1, an ubiquitin protein ligase associated to X-linked intellectual disability and ASD; and a de novo stop variant (p.Q259X) in TPH2, encoding the tryptophan hydroxylase 2 enzyme involved in serotonin synthesis and associated with susceptibility to attention deficit-hyperactivity disorder (ADHD). TPH2, expressed in central and peripheral nervous tissues, modulates various physiological functions, including gut motility and sleep. To the best of our knowledge, this is the first case presenting with ASD, cognitive impairment, sleep, and gastrointestinal disturbances linked to both HUWE1 and TPH2 genes. Our findings could contribute to the existing knowledge on clinical and genetic diagnosis of patients with ASD presentation with comorbidities.

Consensus based recommendations for diagnosis and medical management of Poland syndrome (sequence).

BACKGROUND: Poland syndrome (OMIM: 173800) is a disorder in which affected individuals are born with missing or underdeveloped muscles on one side of the body, resulting in abnormalities that can affect the chest, breast, shoulder, arm, and hand. The extent and severity of the abnormalities vary among affected individuals. MAIN BODY: The aim of this work is to provide recommendations for the diagnosis and management of people affected by Poland syndrome based on evidence from literature and experience of health professionals from different medical backgrounds who have followed for several years affected subjects. The literature search was performed in the second half of 2019. Original papers, meta-analyses, reviews, books and guidelines were reviewed and final recommendations were reached by consensus. CONCLUSION: Being Poland syndrome a rare syndrome most recommendations here presented are good clinical practice based on the consensus of the participant experts.

In-vivo genetic ablation of metabotropic glutamate receptor type 5 slows down disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease due to motor neuron (MN) loss. The mechanisms causing selective MN death are largely unknown, thus prejudicing successful pharmacological treatments. Major causes of MN damage are effects downstream of the abnormal glutamate (Glu) neurotransmission. Group I metabotropic Glu receptors (mGluR1, mGluR5) actively contribute to the excitotoxicity in ALS and represent druggable molecular targets. We previously demonstrated that halving mGluR1 or mGluR5 expression in the widely studied SOD1G93A mouse model of ALS had a positive impact on disease onset, clinical progression and survival, as well as on cellular and biochemical parameters altered in ALS. Whereas these effects were similar in female and male mGluR1 heterozygous SOD1G93Amice, only male mGluR5 heterozygous SOD1G93A mice showed improved motor skills during disease progression. To further validate the role of Group I mGluRs in ALS, we generated in this study mGluR1 or mGluR5 null mice expressing the SOD1G93A mutation (SOD1G93AGrm1crv4/crv4 or SOD1G93AGrm5-/-, respectively). SOD1G93AGrm1crv4/crv4 mice showed early and progressive motor impairments and died even before SOD1G93A mice, while SOD1G93AGrm5-/- mice exhibited delayed disease onset, longer survival, and ameliorated motor skills than SOD1G93A mice. No difference between female and male SOD1G93AGrm5-/- mice were observed. These effects were associated with enhanced MN preservation and decreased astrocytic and microglial activation. Our results strongly support the assumption that constitutively lowering of mGluR5 expression has a positive impact in mice with ALS by counteracting the abnormal Glu transmission and this could be a potentially effective pharmacological target in ALS.

Intragenic duplication of KCNQ5 gene results in aberrant splicing leading to a premature termination codon in a patient with intellectual disability.

The KCNQ5 gene, widely expressed in the brain, encodes a voltage-gated potassium channel (Kv7.5) important for neuronal function. Here, we report a novel KCNQ5 intragenic duplication at 6q13 spanning about 239 Kb of genomic DNA, identified by array comparative genomic hybridization (array-CGH). The duplication was found in heterozygosity in an adult patient affected by mild intellectual disability with history of absence epilepsy in adolescence, with no EEG nor MRI alterations. By in vitro analyses we demonstrated that this copy number variation (CNV) led to an aberrant transcript with exon 2-11 skipping and a premature stop codon causing, most likely, haploinsufficiency. The Kv7.5 channel plays an important role in the regulation of M-type current and afterhyperpolarization conductances which contribute to neuronal excitability. A recently published paper described KCNQ5 missense mutations in individuals with intellectual disability and treatment-resistant epilepsy that were thought to act through either loss-of-function or gain-of-function mechanisms, associated in both cases with altered neuronal excitability. In the case reported here, we showed that no functional protein can be produced from the allele involved by the intragenic duplication. This evidence strongly supports the hypothesis of KCNQ5 haploinsufficiency, which could lead to altered neuronal excitability, thus contributing to seizure susceptibility and intellectual disability.

Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings.

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (±)baclofen (3 µM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1α with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 µM (±)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03-1 µM), inactive on its own on GABA exocytosis, amplified the 3 µM (±)baclofen-induced inhibition of [3H]GABA overflow. The ( ± )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (±)baclofen (1 µM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 µM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (±)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor-receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity.

Poland syndrome: A proposed classification system and perspectives on diagnosis and treatment.

Poland Syndrome (PS) is a rare condition, with an estimated incidence of approximately 1 per 30,000 births and encompasses a wide range of severities of chest and upper arm anomalies. The etiology remains unknown, but genetic involvement is suspected. Few radiological investigations have proven useful in the study PS phenotypes and we propose a reference algorithm for guiding pediatricians. Our experience with 245 PS patients in the last 10 years stimulated a phenotypical classification of PS. The management of the different PS types and a therapeutic algorithm according to the phenotypical features of each PS patient are also proposed.

FGF23 and Fetuin-A Interaction in the Liver and in the Circulation.

Recently it has been demonstrated that Fetuin-A, an anti-inflammatory protein synthesized by the liver, is produced also in bone by an FGF23-regulated pathway. FGF23 has been also demonstrated to induce inflammatory cytokine production in the liver. This study aimed to explore if FGF23 plays a role in the Fetuin-A production in the liver cells too and the possible relationships with FGF23 pro-inflammatory effects. FGF23 and Fetuin-A were studied in liver, kidney and in plasma with immunochemistry, immunoprecipitation, western blot, chromatin immunoprecipitation, duolink, ELISA, qrtPCR methodology. FGF23 is produced, but not secreted by the liver cells. In hepatocytes and circulation, FGF23 was present only strictly linked to Fetuin-A, while Fetuin-A was found also in unbounded form. No link was observed in the kidney. FGF23 up to 600 pg/ml stimulates, while, at higher concentrations, reduces Fetuin-A expression. Notably, overall the range of concentrations, FGF23 stimulates Fetuin-A promoter, TNFα and IL6 expression. In the nucleus, FGF23 seems to act as a direct transcription factor of Fetuin-A promoter. These results suggest that FGF23 played a direct regulatory role in Fetuin-A expression in liver cells with a biphasic effect: Fetuin-A progressively increases when FGF23 increases up to 400-600 pg/mL, and declines at higher FGF23 concentrations. These results lead us to hypothesize: a) a possible epigenetic post-transcriptional regulation; b) a possible counter-regulatory effect of FGF23 induced inflammatory cytokines (TNFα/ NF-κB mechanism). This study could add an additional key for the interpretation of the possible mechanisms linking FGF23, Fetuin-A and inflammation in CKD patients and suggests a role for FGF23 as transcription factor.

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1crv4/crv4) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1crv4 and Grm5ko mice to generate double mutants (Grm1crv4/crv4Grm5ko/ko) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia.

In-vivo effects of knocking-down metabotropic glutamate receptor 5 in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1G93A mice with reduced expression of mGluR5 (SOD1G93AGrm5-/+) by crossing the SOD1G93A mutant mouse with the mGluR5 heterozigous Grm5-/+ mouse. SOD1G93AGrm5-/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1G93AGrm5-/+ mice. Unexpectedly, only male SOD1G93AGrm5-/+ mice showed improved motor skills during disease progression vs. SOD1G93A mice, while SOD1G93AGrm5-/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease.

Phenotypic characterization of Grm1crv4 mice reveals a functional role for the type 1 metabotropic glutamate receptor in bone mineralization.

Recent increasing evidence supports a role for neuronal type signaling in bone. Specifically glutamate receptors have been found in cells responsible for bone remodeling, namely the osteoblasts and the osteoclasts. While most studies have focused on ionotropic glutamate receptors, the relevance of the metabotropic glutamate signaling in bone is poorly understood. Specifically type 1 metabotropic glutamate (mGlu1) receptors are expressed in bone, but the effect of its ablation on skeletal development has never been investigated. Here we report that Grm1crv4/crv4 mice, homozygous for an inactivating mutation of the mGlu1 receptor, and mainly characterized by ataxia and renal dysfunction, exhibit decreased body weight, bone length and bone mineral density compared to wild type (WT) animals. Blood analyses of the affected mice demonstrate the absence of changes in circulating factors, such as vitamin D and PTH, suggesting renal damage is not the main culprit of the skeletal phenotype. Cultures of osteoblasts lacking functional mGlu1 receptors exhibit less homogeneous collagen deposition than WT cells, and present increased expression of osteocalcin, a marker of osteoblast maturation. These data suggest that the skeletal damage is directly linked to the absence of the receptor, which in turn leads to osteoblasts dysfunction and earlier maturation. Accordingly, skeletal histomorphology suggests that Grm1crv4/crv4 mice exhibit enhanced bone maturation, resulting in premature fusion of the growth plate and shortened long bones, and further slowdown of bone apposition rate compared to the WT animals. In summary, this work reveals novel functions of mGlu1 receptors in the bone and indicates that in osteoblasts mGlu1 receptors are necessary for production of normal bone matrix, longitudinal bone growth, and normal skeletal development.

Assessment of copy number variations in 120 patients with Poland syndrome.

BACKGROUND: Poland Syndrome (PS) is a rare congenital disorder presenting with agenesis/hypoplasia of the pectoralis major muscle variably associated with thoracic and/or upper limb anomalies. Most cases are sporadic, but familial recurrence, with different inheritance patterns, has been observed. The genetic etiology of PS remains unknown. Karyotyping and array-comparative genomic hybridization (CGH) analyses can identify genomic imbalances that can clarify the genetic etiology of congenital and neurodevelopmental disorders. We previously reported a chromosome 11 deletion in twin girls with pectoralis muscle hypoplasia and skeletal anomalies, and a chromosome six deletion in a patient presenting a complex phenotype that included pectoralis muscle hypoplasia. However, the contribution of genomic imbalances to PS remains largely unknown. METHODS: To investigate the prevalence of chromosomal imbalances in PS, standard cytogenetic and array-CGH analyses were performed in 120 PS patients. RESULTS: Following the application of stringent filter criteria, 14 rare copy number variations (CNVs) were identified in 14 PS patients in different regions outside known common copy number variations: seven genomic duplications and seven genomic deletions, enclosing the two previously reported PS associated chromosomal deletions. These CNVs ranged from 0.04 to 4.71 Mb in size. Bioinformatic analysis of array-CGH data indicated gene enrichment in pathways involved in cell-cell adhesion, DNA binding and apoptosis processes. The analysis also provided a number of candidate genes possibly causing the developmental defects observed in PS patients, among others REV3L, a gene coding for an error-prone DNA polymerase previously associated with Möbius Syndrome with variable phenotypes including pectoralis muscle agenesis. CONCLUSIONS: A number of rare CNVs were identified in PS patients, and these involve genes that represent candidates for further evaluation. Rare inherited CNVs may contribute to, or represent risk factors of PS in a multifactorial mode of inheritance.

Three-dimensional podocyte-endothelial cell co-cultures: Assembly, validation, and application to drug testing and intercellular signaling studies.

Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies.

Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina.

mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5(-/-) mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

Thrombocytopenia-absent radius (TAR) syndrome due to compound inheritance for a 1q21.1 microdeletion and a low-frequency noncoding RBM8A SNP: a new familial case.

BACKGROUND: Thrombocytopenia-absent radius syndrome (TAR; MIM 274000) is a rare autosomal recessive disorder combining specific skeletal abnormalities with a reduced platelet count. TAR syndrome has been associated with the compound inheritance of an interstitial microdeletion in 1q21.1 and a low frequency noncoding RBM8A SNP. RESULTS: Here, we report on a patient with scapulo-humeral hypoplasia, bilateral radio-ulnar agenesis with intact thumbs, bilateral proximal positioning of the first metacarpal, bilateral fifth finger clinodactyly, bilateral radial deviation of the hands, and thrombocytopenia. Molecular studies showed compound heterozygosity for the 1q21.1 microdeletion and the RBM8A rs139428292 variant in hemizygous state, inherited from the father and the mother, respectively. A second aborted fetus presented TAR features and 1q21.1 microdeletion. DISCUSSION: The complex inheritance pattern resulted in reduced expression of Y14, the protein encoded by RBM8A, and a component of the core exon-junction complex (EJC) in platelets. Further studies are needed to explain how Y14 insufficiency and subsequent defects of the EJC could cause the skeletal, haematological and additional features of TAR syndrome. In this study, we discuss other factors that could influence the overall phenotype of patients affected by TAR syndrome. CONCLUSION: In this study, we discuss other factors that could influence the overall phenotype of patients affected by TAR syndrome.

Clinical and molecular characterization of a patient with interstitial 6q21q22.1 deletion.

BACKGROUND: Interstitial 6q deletions, involving the 6q15q25 chromosomal region, are rare events characterized by variable phenotypes and no clear karyotype/phenotype correlation has been determined yet. RESULTS: We present a child with a 6q21q22.1 deletion, characterized by array-CGH, associated with developmental delay, intellectual disability, microcephaly, facial dysmorphisms, skeletal, muscle, and brain anomalies. DISCUSSION: In our patient, the 6q21q22.1 deleted region contains ten genes (TRAF3IP2, FYN, WISP3, TUBE1, LAMA4, MARCKS, HDAC2, HS3ST5, FRK, COL10A1) and two desert gene regions. We discuss here if these genes had some role in determining the phenotype of our patient in order to establish a possible karyotype/phenotype correlation.