Gap junctions or hemichannel-dependent and independent roles of connexins in cataractogenesis and lens development

In the last decade or so, increasing evidences suggest that the mutations of two connexin genes, GJA3 and GJA8, are directly linked to human congenital cataracts in North and Central America, Europe and Asia. GIA3 and GIA8 genes encode gap junction-forming proteins, connexin (Cx) 46 and Cx50, respectively. These two connexins are predominantly expressed in lens fiber cells. Majority of identified mutations are missense, and the mutated sites are scattered across various domains of connexin molecules. Genetic deletion of either of these two genes leads to the development of cataracts; however, the types of cataracts developed are distinctive. More interestingly, microphthalmia is only developed in Cx50, but not Cx46 deficient mice, suggesting the unique role of Cx50 in lens cell growth and development. Knockin studies with the replacement of Cx46 or Cx50 at their respective gene locus further demonstrate the unique properties of these two connexins. Furthermore, the function of Cx50 in epithelial-fiber differentiation appears to be independent of its conventional role in forming gap junction junction channels. Due to their specific functions in maintaining lens clarity and development, and their malfunctions resulting in lens cataractogenesis and developmental impairment, connexin molecules could be developed as potential drug targets for therapeutic intervention for treatment of cataracts and other eye disorders. Recent advances in basic research of lens connexins and the discoveries of clinical disorders as a result of lens connexin dysfunctions are summarized and discussed here.     

Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens.

Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.     

TSR1突变导致先天性白内障及其在晶状体中的表达

先天性白内障(congenital cataract,CC)是一种罕见的晶状体发育异常疾病,主要表现为晶状体部分或完全浑浊。先天性白内障遗传异质性高,已鉴定的致病基因多达266个。本研究在一个中国先天性白内障家系中通过全基因组测序及Sanger测序验证,筛查到一个新的先天性白内障候选致病基因TSR1,与家系疾病表型共分离。通过minigene实验证实该变异影响TSR1基因mRNA剪接。Western blotting、免疫荧光和RT-PCR实验证实TSR1在人晶状体上皮细胞SRA01/04、年龄相关性白内障患者晶状体前囊膜组织、24周人胎眼晶状体和小鼠晶状体中表达。通过对iSyTE数据库的分析发现,Tsr1在小鼠的胚胎期和不同发育时期的晶状体中都有表达,且在晶状体特异性CBP:p300双敲除小鼠中Tsr1表达下调。提取在CBP:p300双敲除小鼠晶状体中与Tsr1具有相同表达模式的一组基因进行蛋白质-蛋白质相互作用网络(protein-protein interaction,PPI)分析,结果表明筛选出6个基因与Tsr1存在直接相互作用。GO功能分析表明Tsr1参与核糖体的组装,还可能在MAPK-Erk信号通路中发挥作用,为进一步明确Tsr1在晶状体中的功能提供了有价值的研究线索。     

Crystallins and hereditary cataracts: molecular mechanisms and potential for therapy

Hereditary childhood cataracts can arise from single-point mutations in genes encoding crystallins, the major protein components of the lens. The cataracts are most commonly inherited by an autosomal dominant mechanism. The nature of the changes in the lens resulting from these point mutations in crystallin genes has not been fully characterised. While aggregation and light scattering associated with expression of the mutant crystallin protein may be an end point, it is also necessary to determine the progression of changes induced at the level of development and differentiation. A key finding in recent work is that cell death or cytotoxicity is associated with mutations in alpha A-crystallin. The variable morphology or localisation of the cataract in different pedigrees, even with the identical crystallin gene mutation, has led to the idea that other environmental or genetic factors interact to give the final lens phenotype. The study of mechanisms of formation of hereditary cataracts may lead to a greater understanding of the mechanisms that lead to age-related cataracts, a very common cause of blindness in the ageing population.     

Zebrafish models for the functional genomics of neurogenetic disorders

In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Molecular genetics of retinitis pigmentosa.

Retinitis pigmentosa is a model for the study of genetic diseases. Its genetic heterogeneity is reflected in the different forms of inheritance (autosomal dominant, autosomal recessive, or X-linked) and, in a few families, in the presence of mutations in the visual pigment rhodopsin. Clinical and molecular genetic studies of these disorders are discussed. Animal models of retinal degeneration have been investigated for many years with the hope of gaining insight into the cause of photoreceptor cell death. Recently, the genes responsible for two of these animal disorders, the rds and rd mouse genes, have been isolated and characterized. The retinal degeneration of the rd mouse is presented in detail. The possible involvement of human analogues of these mouse genes in human retinal diseases is being investigated.     

Congenital cataracts and their molecular genetics

Cataract can be defined as any opacity of the crystalline lens. Congenital cataract is particularly serious because it has the potential for inhibiting visual development, resulting in permanent blindness. Inherited cataracts represent a major contribution to congenital cataracts, especially in developed countries. While cataract represents a common end stage of mutations in a potentially large number of genes acting through varied mechanisms in practice most inherited cataracts have been associated with a subgroup of genes encoding proteins of particular importance for the maintenance of lens transparency and homeostasis. The increasing availability of more detailed information about these proteins and their functions and is making it possible to understand the pathophysiology of cataracts and the biology of the lens in general.     

Diverse requirements for Notch signalling in mammals

The Notch signalling pathway has a central role in a wide variety of developmental processes and it is not therefore surprising that mutations in components of this pathway can cause dramatic human genetic disorders. One developmental process in which the Notch pathway is involved at multiple levels is somitogenesis, the mechanism by which the embryo is divided into segments that ultimately form structures such as the axial skeleton and skeletal muscle of the trunk. We are investigating the human genetic disorder spondylocostal dysplasia (SCD), which is a group of malsegmentation syndromes that occur when this process is disrupted. Mutations in the Notch ligand DELTA-LIKE 3 (DLL3) are responsible for cases of autosomal recessive SCD type I (SCDO1), and we are using information derived from these mutations to study the structure of the DLL3 protein. To aid in elucidation of the underlying developmental defect in SCDO1, we have generated a mouse model by targeted deletion of the Dll3 gene (Dunwoodie et al., 2002). These mice show segmentation defects similar to those seen in SCDO1. In addition, these mice have a distinct set of neural defects that may be useful in future neurological assessment of affected individuals. Finally, since not all cases of SCD are due to mutation of DLL3, we are investigating various genes to find other candidates involved in this genetic disease.     

Organization and annotation of the Xcat critical region: elimination of seven positional candidate genes

Xcat mice display X-linked congenital cataracts and are a mouse model for the human X-linked cataract disease Nance Horan syndrome (NHS). The genetic defect in Xcat mice and NHS patients is not known. We isolated and sequenced a BAC contig representing a portion of the Xcat critical region. We combined our sequencing data with the most recent mouse sequence assemblies from both Celera and public databases. The sequence of the 2.2-Mb Xcat critical region was then analyzed for potential Xcat candidate genes. The coding regions of the seven known genes within this area (Rai2, Rbbp7, Ctps2, Calb3, Grpr, Reps2, and Syap1) were sequenced in Xcat mice and no mutations were detected. The expression of Rai2 was quantitatively identical in wild-type and Xcat mutant eyes. These results indicate that the Xcat mutation is within a novel, undiscovered gene.     

Ionizing radiation and genetic risks. X. The potential "disease phenotypes" of radiation-induced genetic damage in humans: perspectives from human molecular biology and radiation genetics.

Estimates of genetic risks of radiation exposure of humans are traditionally expressed as expected increases in the frequencies of genetic diseases (single-gene, chromosomal and multifactorial) over and above those of naturally-occurring ones in the population. An important assumption in expressing risks in this manner is that gonadal radiation exposures can cause an increase in the frequency of mutations and that this would result in an increase in the frequency of genetic diseases under study. However, despite compelling evidence for radiation-induced mutations in experimental systems, no increases in the frequencies of genetic diseases of concern or other adverse effects (i.e., those which are not formally classified as genetic diseases), have been found in human studies involving parents who have sustained radiation exposures. The known differences between spontaneous mutations that underlie naturally-occurring single-gene diseases and radiation-induced mutations studied in experimental systems now permit us to address and resolve these issues to some extent. The fact that spontaneous mutations (among which are point mutations and DNA deletions generally restricted to the gene) originate through a number of different mechanisms and that the latter are intimately related to the DNA organization of the genes, are now well-documented. Further, spontaneous mutations include those that cause diseases through loss of function as well as gain of function of genes. In contrast, most radiation-induced mutations studied in experimental systems (although identified through the phenotypes of the marker genes) are predominantly multigene deletions which cause loss of function; the recoverability of an induced deletion in a livebirth seems dependent on whether the gene and the genomic region in which it is located can tolerate heterozygosity for the deletion and yet be compatible with viability. In retrospect, the successful mutation test systems (such as the mouse specific locus test) used in radiation studies have involved genes which are non-essential for survival and are also located in genomic regions, likewise non-essential for survival. In contrast, most of the human genes at which induced mutations have been looked for, do not seem to have these attributes. The inference therefore is that the failure to find induced germline mutations in humans is not due to the resistance of human genes to induced mutations but due to the structural and functional constraints associated with their recoverability in livebirths. Since the risk of inducible genetic diseases in humans is estimated using rates of "recovered" mutations in mice, there is a need to introduce appropriate correction factors to bridge the gap between these rates and the rates at which mutations causing diseases are potentially recoverable in humans. Since the whole genome is the "target" for radiation-induced genetic damage, the failure to find increases in the frequencies of specific single-gene diseases of societal concern does not imply that there are no genetic risks of radiation exposures: the problem lies in delineating the phenotypes of recoverable genetic damage that are recognizable in livebirths. Data from studies of naturally-occurring microdeletion syndromes in humans and those from mouse radiation studies are instructive in this regard. They (i) support the view that growth retardation, mental retardation and multisystem developmental abnormalities are likely to be among the quantitatively more important adverse effects of radiation-induced genetic damage than mutations in a few selected genes and (ii) underscore the need to expand the focus in risk estimation from known genetic diseases (as has been the case thus far) to include these induced adverse developmental effects although most of these are not formally classified as "genetic diseases". (ABSTRACT TRUNCATED)     

Models and assumptions underlying genetic risk assessment

Various methods employed for estimating the genetic risks of radiation are reviewed. With the doubling-dose method, genetic damage is expressed as an increase in cases of known genetic disease. The actual doubling dose is based on figures obtained with the mouse. There have been no recent data on induced mutation frequencies. Recent results suggest that the prevalence figure for multifactorial disease may be at least one order of magnitude higher than before. Various assumptions underlying the doubling-dose concept are discussed in the light of recent findings on: (1) spontaneous mutations resulting from insertion elements, and (2) the comparability between spontaneous and induced mutations. The so-called direct method makes use of figures for induction of dominant mutations affecting the skeleton and the lens of the eye in the mouse, and of translocation induction in monkeys. Induction rates are converted to overall rates of induced dominant effects in man by applying certain assumptions. The proportionality between dose and effect is the basis for all genetic risk assessments. The possible significance of data on human lymphocytes indicating a threshold below 4 rad and the induction of repair enzymes by low radiation doses is discussed. The parallelogram approach is based on the principle that estimates can be obtained on the amount of genetic damage that cannot always be assessed directly. Thus mutations in mouse germ cells can be predicted by using mutation frequencies in cultured mammalian cells and O6-ethylguanine adducts. Measurement of haemoglobin mutations in human and mouse erythrocytes, and of HPRT-deficient mutations in lymphocytes of man and mouse should make more precise estimates of mutation frequencies in human germ cells possible. The development of a database on mutations in somatic cells of the mouse, their induction frequencies and molecular nature are considered an important priority. Used in combination with mouse germ-cell mutation frequencies, they should enable more precise risk estimates on the basis of mutations in somatic cells of man.     

Identifying novel genes for atherosclerosis through mouse-human comparative genetics

Susceptibility to atherosclerosis is determined by both environmental and genetic factors. Its genetic determinants have been studied by use of quantitative-trait-locus (QTL) analysis. So far, 21 atherosclerosis QTLs have been identified in the mouse: 7 in a high-fat-diet model only, 9 in a sensitized model (apolipoprotein E- or LDL [low-density lipoprotein] receptor-deficient mice) only, and 5 in both models, suggesting that different gene sets operate in each model and that a subset operates in both. Among the 27 human atherosclerosis QTLs reported, 17 (63%) are located in regions homologous (concordant) to mouse QTLs, suggesting that these mouse and human atherosclerosis QTLs have the same underlying genes. Therefore, genes regulating human atherosclerosis will be found most efficiently by first finding their orthologs in concordant mouse QTLs. Novel mouse QTL genes will be found most efficiently by using a combination of the following strategies: identifying QTLs in new crosses performed with previously unused parental strains; inducing mutations in large-scale, high-throughput mutagenesis screens; and using new genomic and bioinformatics tools. Once QTL genes are identified in mice, they can be tested in human association studies for their relevance in human atherosclerotic disease.     

What transgenic mice tell us about neurodegenerative disease

The recent broad advance in our understanding of human neurodegenerative diseases is based on the application of a new molecular approach. Through linkage analysis, the genes responsible for Huntington's disease, the spinocerebellar ataxias, and familial forms of Alzheimer's disease and amyotrophic lateral sclerosis (ALS) have been identified and cloned. The characterization of pathogenic mutations in such genes allows the creation of informative transgenic mouse models as, without exception, the genetic forms of adult neurodegenerative disease are due to toxicity of the mutant protein. Transgenic models provide insight into the oxidative mechanisms in ALS pathogenesis, the pathogenicity of expanded polyglutamine tracts in CAG triplet repeat disorders, and amyloidogenesis in Alzheimer's disease. Although such models have their limitations, they currently provide the best entry point for the study of human neurodegenerative diseases.     

Role of cilia in structural birth defects: Insights from ciliopathy mutant mouse models

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia‐related genes from many large‐scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs. Birth Defects Research (Part C) 102:115–125, 2014. © 2014 Wiley Periodicals, Inc.