Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Differential Contributions of Vision,Touch and Muscle Proprioception to the Coding of Hand Movements

To further elucidate the mechanisms underlying multisensory integration, this study examines the controversial issue of whether congruent inputs from three different sensory sources can enhance the perception of hand movement. Illusory sensations of clockwise rotations of the right hand were induced by either separately or simultaneously stimulating visual, tactile and muscle proprioceptive channels at various intensity levels. For this purpose, mechanical vibrations were applied to the pollicis longus muscle group in the subjects’ wrists, and a textured disk was rotated under the palmar skin of the subjects’ right hands while a background visual scene was projected onto the rotating disk. The elicited kinaesthetic illusions were copied by the subjects in real time and the EMG activity in the adductor and abductor wrist muscles was recorded. The results show that the velocity of the perceived movements and the amplitude of the corresponding motor responses were modulated by the nature and intensity of the stimulation. Combining two sensory modalities resulted in faster movement illusions, except for the case of visuo-tactile co-stimulation. When a third sensory input was added to the bimodal combinations, the perceptual responses increased only when a muscle proprioceptive stimulation was added to a visuo-tactile combination. Otherwise, trisensory stimulation did not override bimodal conditions that already included a muscle proprioceptive stimulation. We confirmed that vision or touch alone can encode the kinematic parameters of hand movement, as is known for muscle proprioception. When these three sensory modalities are available, they contribute unequally to kinaesthesia. In addition to muscle proprioception, the complementary kinaesthetic content of visual or tactile inputs may optimize the velocity estimation of an on-going movement, whereas the redundant kinaesthetic content of the visual and tactile inputs may rather enhance the latency of the perception.     

Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis

The Ret receptor tyrosine kinase mediates physiological signals of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and is essential for postnatal survival in mice. It is implicated in a number of human diseases and developmental abnormalities. Here, we describe our analyses of mice expressing a Ret mutant (RetDN) with diminished kinase activity that inhibits wild-type Ret activity, including its activation of AKT. All RetDN/+ mice died by 1 month of age and had distal intestinal aganglionosis reminiscent of Hirschsprung disease (HSCR) in humans. The RetDN/+ proximal small intestine also had severe hypoganglionosis and reduction in nerve fiber density, suggesting a potential mechanism for the continued gastric dysmotility in postsurgical HSCR patients. Unlike Ret-null mice, which have abnormalities in the parasympathetic and sympathetic nervous systems, the RetDN/+ mice only had defects in the parasympathetic nervous system. A small proportion of RetDN/+ mice had renal agenesis, and the remainder had hypoplastic kidneys and developed tubulocystic abnormalities postnatally. Postnatal analyses of the testes revealed a decreased number of germ cells, degenerating seminiferous tubules, maturation arrest and apoptosis, indicating a crucial role for Ret in early spermatogenesis.     

A Robust GWSS Method to Simultaneously Detect Rare and Common Variants for Complex Disease

The rapid advances in sequencing technologies and the resulting next-generation sequencing data provide the opportunity to detect disease-associated variants with a better solution, in particular for low-frequency variants. Although both common and rare variants might exert their independent effects on the risk for the trait of interest, previous methods to detect the association effects rarely consider them simultaneously. We proposed a class of test statistics, the generalized weighted-sum statistic (GWSS), to detect disease associations in the presence of common and rare variants with a case-control study design. Information of rare variants was aggregated using a weighted sum method, while signal directions and strength of the variants were considered at the same time. Permutations were performed to obtain the empirical p-values of the test statistics. Our simulation showed that, compared to the existing methods, the GWSS method had better performance in most of the scenarios. The GWSS (in particular VDWSS-t) method is particularly robust for opposite association directions, association strength, and varying distributions of minor-allele frequencies. It is therefore promising for detecting disease-associated loci. For empirical data application, we also applied our GWSS method to the Genetic Analysis Workshop 17 data, and the results were consistent with the simulation, suggesting good performance of our method. As re-sequencing studies become more popular to identify putative disease loci, we recommend the use of this newly developed GWSS to detect associations with both common and rare variants.     

The Human Phenotype Ontology: Semantic Unification of Common and Rare Disease

The Human Phenotype Ontology (HPO) is widely used in the rare disease community for differential diagnostics, phenotype-driven analysis of next-generation sequence-variation data, and translational research, but a comparable resource has not been available for common disease. Here, we have developed a concept-recognition procedure that analyzes the frequencies of HPO disease annotations as identified in over five million PubMed abstracts by employing an iterative procedure to optimize precision and recall of the identified terms. We derived disease models for 3,145 common human diseases comprising a total of 132,006 HPO annotations. The HPO now comprises over 250,000 phenotypic annotations for over 10,000 rare and common diseases and can be used for examining the phenotypic overlap among common diseases that share risk alleles, as well as between Mendelian diseases and common diseases linked by genomic location. The annotations, as well as the HPO itself, are freely available.     

A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs

The discovery that RNA viruses, lacking any DNA intermediate, can be engineered to express both coding and noncoding RNAs suggests that this platform may have therapeutic value as a delivery vehicle. Here we illustrate that a self-replicating, noninfectious RNA, modeled on influenza virus, provides one such example of a versatile in vivo delivery system for silencing and/or expressing a desired RNA for therapeutic purposes.     

编码序列和非编码序列的3-tuple分布特征

非编码序列,特别是内含子的起源,是一个重要的悬而未决的问题。首先通过计算模式生物的编码序列和非编码序列的不同阅读框中3-tupie的频率分布,发现编码区中不同阅读框具有十分不同的3-tuple分布,而在非编码区中,不同阅读框的3-tuple分布几乎相等,并且这一性质不具有物种依赖性。为了描述分布差异的程度,引进夏量一对称相对熵,并通过比较原核生物和真核生物,发现无论是编码区还是非编码区,原核生物都具有比真核生物更高的SRE值。进一步研究表明,某一生物的SRE值与该生物全基因组中编码区所占的百分比存在一定的相关性（相关系数为0．86）。计算机模拟进化实验发现,2％的突变就足以使典型的嗯核生物编码区高SRE值变为真核生物内含子区特有的低SRE值。比对数据库中已经注释的内含子和编码区序列,证明确实有一部分与编码区具有很高同源性的内含子序列。实验表明,至少部分真核生物的内含子可能起源于编码序列,同时也说明SRE可能被用于研究物种基因组序列的进化。     

Glial Contributions to Neural Function and Disease

The nervous system consists of neurons and glial cells. Neurons generate and propagate electrical and chemical signals, whereas glia function mainly to modulate neuron function and signaling. Just as there are many different kinds of neurons with different roles, there are also many types of glia that perform diverse functions. For example, glia make myelin; modulate synapse formation, function, and elimination; regulate blood flow and metabolism; and maintain ionic and water homeostasis to name only a few. Although proteomic approaches have been used extensively to understand neurons, the same cannot be said for glia. Importantly, like neurons, glial cells have unique protein compositions that reflect their diverse functions, and these compositions can change depending on activity or disease. Here, I discuss the major classes and functions of glial cells in the central and peripheral nervous systems. I describe proteomic approaches that have been used to investigate glial cell function and composition and the experimental limitations faced by investigators working with glia.The nervous system is composed of neurons and glial cells that function together to create complex behaviors. Traditionally, glia have been considered to be merely passive contributors to brain function, resulting in a pronounced neurocentric bias among neuroscientists. Some of this bias reflects a paucity of knowledge and tools available to study glia. However, this view is rapidly changing as new tools, model systems (culture and genetic), and technologies have permitted investigators to show that glia actively sculpt and modulate neuronal properties and functions in many ways. Glia have been thought to outnumber neurons by 10:1, although more recent studies suggest the ratio in the human brain is closer to 1:1 with region-specific differences (1). There are many different types of glia, some of which are specific to the central nervous system (CNS),¹ whereas others are found only in the peripheral nervous system (PNS). The main types of CNS glia include astrocytes, oligodendrocytes, ependymal cells, radial glia, and microglia. In the PNS, the main glial cells are Schwann cells, satellite cells, and enteric glia. These cells differ and are classified according to their morphologies, distinct anatomical locations in the nervous system, functions, developmental origins, and unique molecular compositions. Among the different classes of glia there are additional subclasses that reflect further degrees of specialization. In this review, I will discuss the characteristics and functions of the major glial cell types including astrocytes, microglia, and the myelin-forming oligodendrocytes (CNS) and Schwann cells (PNS). Because of space limitations, it is impossible to give a complete accounting of all glia and what is known about each of these cell types. Therefore, I encourage the interested reader to refer to some of the many excellent reviews referenced below that focus on individual glial cell types. Finally, I will discuss proteomic studies of glial cell function and some of the unique challenges investigators face when working with these cells.     

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

Introduction

In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI.

Patient and Methods

Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis.

Results

Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively.

Conclusion

One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival.     

Noncoding Mutations of HGF Are Associated with Nonsyndromic Hearing Loss, DFNB39

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3′ untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.     

Common genetic variants in GAL,GAP43 and NRSN1 and interaction networks confer susceptibility to Hirschsprung disease

Hirschsprung disease (HSCR) is a severe multifactorial genetic disorder. Microarray studies indicated GAL, GAP43 and NRSN1 might contribute to the altered risk in HSCR. Thus, we focused on genetic variations in GAL, GAP43 and NRSN1, and the gene‐gene interactions involved in HSCR susceptibility. We recruited a strategy combining case‐control study and MassArray system with interaction network analysis. For GAL, GAP43 and NRSN1, a total of 18 polymorphisms were assessed in 104 subjects with sporadic HSCR and 151 controls of Han Chinese origin. We found statistically significant differences between HSCR and control groups at 5 genetic variants. For each gene, the haplotypes combining all polymorphisms were the most significant. Based on SNPsyn, MDR and GeneMANIA analyses, we observed significant gene‐gene interactions among GAL, GAP43, NRSN1 and our previous identified RELN, GABRG2 and PTCH1. Our study for the first time indicates that genetic variants within GAL, GAP43 and NRSN1 and related gene‐gene interaction networks might be involved in the altered susceptibility to HSCR in the Han Chinese population, which might shed more light on HSCR pathogenesis.     

Laminar and Temporal Expression Dynamics of Coding and Noncoding RNAs in the Mouse Neocortex

1. Download : Download full-size image

     

microRNA和长链非编码RNA在呼吸系统疾病中的表达

非编码RNA包括microRNA(miRNA)和长链非编码RNA(1ncRNA),广泛存在于真核生物的基因组中,通过调控转译过程影响各种生物学效应,在多种生物体的重要生命过程中具有不可替代的作用。研究已证实呼吸系统疾病中miRNA存在差异性表达,而lncRNA可调节分子表型,两者的相互作用机制仍需大量实验进行研究。非编码RNA作为一种新型治疗途径的靶点,其具体作用机制及如何更好地应用于临床值得深入探讨。     

Identification of Genetic Loci Affecting the Severity of Symptoms of Hirschsprung Disease in Rats Carrying Ednrbsl Mutations by Quantitative Trait Locus Analysis

Hirschsprung’s disease (HSCR) is a congenital disease in neonates characterized by the absence of the enteric ganglia in a variable length of the distal colon. This disease results from multiple genetic interactions that modulate the ability of enteric neural crest cells to populate developing gut. We previously reported that three rat strains with different backgrounds (susceptible AGH-Ednrb^sl/sl, resistant F344-Ednrb^sl/sl, and LEH-Ednrb^sl/sl) but the same null mutation of Ednrb show varying severity degrees of aganglionosis. This finding suggests that strain-specific genetic factors affect the severity of HSCR. Consistent with this finding, a quantitative trait locus (QTL) for the severity of HSCR on chromosome (Chr) 2 was identified using an F₂ intercross between AGH and F344 strains. In the present study, we performed QTL analysis using an F₂ intercross between the susceptible AGH and resistant LEH strains to identify the modifier/resistant loci for HSCR in Ednrb-deficient rats. A significant locus affecting the severity of HSCR was also detected within the Chr 2 region. These findings strongly suggest that a modifier gene of aganglionosis exists on Chr 2. In addition, two potentially causative SNPs (or mutations) were detected upstream of a known HSCR susceptibility gene, Gdnf. These SNPs were possibly responsible for the varied length of gut affected by aganglionosis.