Widening of the genetic and clinical spectrum of Lamb-Shaffer syndrome, a neurodevelopmental disorder due to SOX5 haploinsufficiency

Purpose Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved. Methods Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types ofSOX5 alterations. Functional consequences of selected substitutions were investigated. Results Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated. Conclusions This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features

Genome engineering with TALE and CRISPR systems in neuroscience

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience

The endocannabinoid gene <i>faah2a</i> modulates stress-associated behavior in zebrafish

<div><p>The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (<i>abhd4</i>, <i>gde1</i>, <i>napepld)</i>, enzymes that degrade (<i>faah</i>, <i>faah2a</i>, <i>faah2b</i>), and receptors that bind (<i>cnr1</i>, <i>cnr2</i>, <i>gpr55-like</i>) endocannabinoids are expressed in zebrafish (<i>Danio rerio</i>). These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in <i>cnr1</i> and <i>faah2a</i> to test the role of these genes in modulating stress-associated behavior. We showed that disruption of <i>cnr1</i> potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of <i>faah2a</i> attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.</p></div

<i>faah2a</i> modulates stress-associated behavior.

<p>(A) The rolling means of distances travelled by wild type (WT), heterozygous <i>faah2a</i> (HET) mutant, and homozygous <i>faah2a</i> (HOM) mutant zebrafish. The pre-treatment baseline locomotor activity was recorded from −15–0 min, and the post-treatment locomotor activity was recorded from 0–31 min. At time 0 the zebrafish were treated with either E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity at each second is represented as a mean of the distance travelled during the preceding 60 s. (B) The means of distances travelled after the zebrafish were treated with E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity of each group is represented as a mean of the distance travelled per min during the 5–25 min time bin ± 95% CI. Groups with all different letters above the columns are statistically different from each other, while groups with a conserved letter above the columns are not statistically different from each other (Tukey's honest significant difference test, <i>P <</i> 0.05).</p

TALEN-mediated mutagenesis of <i>faah2a</i>.

<p>(A) The <i>faah2a</i> TALEN target site was designed in exon 3 so that mutagenesis would disrupt all predicted splice variants. NCBI Accessions: Gene ID, 436973; DNA, NC_007112.6; mRNA (i), NM_001002700.2; Protein (i), NP_001002700.1. The target site is just upstream of the sequence encoding a lysine in the predicted serine 228 (S) / serine 204 (S) / lysine 129 (K) catalytic triad. (B) An alignment of wild type and mutant <i>faah2a</i> sequences reveals the TALEN-induced indel in the target BsrI restriction enzyme site.</p

Temporal expression patterns of eCB receptor genes.

<p>The time points on all graphs are represented as means ± 95% CI (0.25–1 dpf: 20 larvae/n, n = 3; 2–7 dpf: 10 larvae/n, n = 3). * Indicates that a group is significantly different from the 5 dpf group (Sidak's multiple comparisons test, <i>P <</i> 0.05). (A) The fold change of <i>cnr1</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (B) The fold change of <i>cnr2</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (C) The fold change of <i>loc793909</i> transcript levels relative to 5 dpf as determined by qRT-PCR.</p

<i>cnr1</i> modulates stress-associated behavior.

<p>(A) The rolling means of distances travelled by wild type (WT), heterozygous <i>cnr1</i> (HET) mutant, and homozygous <i>cnr1</i> (HOM) mutant zebrafish. The pre-treatment baseline locomotor activity was recorded from −15–0 min, and the post-treatment locomotor activity was recorded from 0–31 min. At time 0 the zebrafish were treated with either E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity at each second is represented as a mean of the distance travelled during the preceding 60 s. (B) The means of distances travelled after the zebrafish were treated with E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity of each group is represented as a mean of the distance travelled per min during the 5–25 min time bin ± 95% CI. Groups with all different letters above the columns are statistically different from each other, while groups with a conserved letter above the columns are not statistically different from each other (Tukey's honest significant difference test, <i>P <</i> 0.05).</p

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1

International audienceUpon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process

PCNT point mutations and familial intracranial Aneurysms

Objective To identify novel genes involved in the etiology of intracranial aneurysms (IAs) or subarachnoid hemorrhages (SAHs) using whole-exome sequencing. Methods We performed whole-exome sequencing in 13 individuals from 3 families with an autosomal dominant IA/SAH inheritance pattern to look for candidate genes for disease. In addition, we sequenced PCNT exon 38 in a further 161 idiopathic patients with IA/SAH to find additional carriers of potential pathogenic variants. Results We identified 2 different variants in exon 38 from the PCNT gene shared between affected members from 2 different families with either IA or SAH (p.R2728C and p.V2811L). One hundred sixty-four samples with either SAH or IA were Sanger sequenced for the PCNT exon 38. Five additional missense mutations were identified. We also found a second p.V2811L carrier in a family with a history of neurovascular diseases. Conclusion The PCNT gene encodes a protein that is involved in the process of microtubule nucleation and organization in interphase and mitosis. Biallelic loss-of-function mutations in PCNT cause a form of primordial dwarfism (microcephalic osteodysplastic primordial dwarfism type II), and ≈50% of these patients will develop neurovascular abnormalities, including IAs and SAHs. In addition, a complete Pcnt knockout mouse model (Pcnt-/-) published previously showed general vascular abnormalities, including intracranial hemorrhage. The variants in our families lie in the highly conserved PCNT protein-protein interaction domain, making PCNT a highly plausible candidate gene in cerebrovascular disease