The Role of Transposons in Epigenetic Regulation of Ontogenesis

A new insight into the mechanisms underlying implementation of genomic information in the individual development of eukaryotes through interactions of transposons with epigenetic factors dynamically changing during each cell division is described. These mechanisms of stepwise implementation of individual genetic information with characteristic stage- and tissue-specific features in the activities of certain mobile genetic element families are evolutionarily fixed at the species level. In addition, the individual differences caused by their “unscheduled” transpositions can significantly change the regulatory network of the genome altering the phenotype. These changes in individual development can bring about new traits leading to either a disease or better fitness and represent an important component of the variation for natural selection in evolution. A large part of the eukaryotic transposons is altered by mutations and used for formation of the regulatory gene network, changes in the protein-coding genes, and emergence of new nonprotein-coding genes. When inserted into new loci, mobile genetic elements form the basis for microRNA and the domain structures of long noncoding RNA, responding to various types of stress; this is reflected in the specific features of individual development and contributes to variation. The epigenetic factors, including noncoding RNA, DNA methylation, and histone modifications, are tightly associated with mobile genetic elements. The specific features in transposon location in individuals that have emerged owing to spontaneous mutations or those caused by stress impacts can considerably change the interactions in gene networks. This influences the likelihood of survival under changing environmental conditions and reflects a distinct interrelation between the mechanisms of individual development and evolution. There is a parallelism between the mechanisms underlying the rearrangements of genomes caused by transposons in evolution and in individual development. In particular, the responsiveness of transposons to external and internal (microenvironment) factors forms the background for evolutionary construction of transposon-mediated tissue-specific activation patterns of certain transposons during each cell division, which leads to maturation of a reproductive organism. This mechanism is based on tight stage- and tissuespecific interrelation between transposons, epigenetic factors, and protein-coding genes.     

A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice

Polydactyly is a common malformation of vertebrate limbs. In humans a major locus for nonsyndromic pre-axial polydactyly (PPD) has been mapped previously to 7q36. The mouse Hemimelic extra-toes (Hx) mutation maps to a homologous chromosome segment and has been proposed to affect a homologous gene. To understand the molecular changes underlying PPD, we used a positional cloning approach to identify the gene or genes disrupted by the Hx mutation and a closely linked limb mutation, Hammertoe (Hm). High resolution genetic mapping identified a small candidate interval for the mouse mutations located 1.2 cM distal to the Shh locus. The nonrecombinant interval was completely cloned in bacterial artificial chromosomes and searched for genes using a combination of exon trapping, sample sequencing, and mapping of known genes. Two novel genes, Lmbr1 and Lmbr2, are entirely within the candidate interval we defined genetically. The open reading frame of both genes is intact in mutant mice, but the expression of the Lmbr1 gene is dramatically altered in developing limbs of Hx mutant mice. The correspondence between the spatial and temporal changes in Lmbr1 expression and the embryonic onset of the Hx mutant phenotype suggests that the mouse Hx mutation may be a regulatory allele of Lmbr1. The human ortholog of Lmbr1 maps within the recently described interval for human PPD, strengthening the possibility that both mouse and human limb abnormalities are due to defects in the same highly conserved gene.     

Seizure disorders in mutant mice: relevance to human epilepsies.

The rate at which mutant genes producing an epileptic phenotype in mice have been identified over the past few years has been astounding. Manipulating the genome of mice has led to identification of a diversity of genes whose absence or modification either causes epileptic seizures or, conversely, limits epileptogenesis. In addition, positional cloning of genes in which spontaneously arising mutations cause epilepsy in mice has led to the identification of genes encoding voltage- and ligand-gated ion channels. Finally, engineering a mutation that mimics a rare form of human epilepsy has led to a mouse line with a phenotype similar to that of the human disease. Taken together, these discoveries promise to shed light on the mechanisms underlying genetic control of neuronal excitability, suggest candidate genes underlying genetic forms of human epilepsy, and provide a valuable model with which to elucidate how the genotype produces the phenotype of a rare form of human epilepsy.     

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.     

Pleiotropic effects of pigmentation genes in horses

Horses are valued for the beauty and variety of colouration and coat patterning. To date, eleven different genes have been characterized that contribute to the variation observed in the horse. Unfortunately, mutations involving pigmentation often lead to deleterious effects in other systems, some of which have been described in the horse. This review focuses on six such pleiotropic effects or associations with pigmentation genes. These include neurological defects (lethal white foal syndrome and lavender foal syndrome), hearing defects, eye disorders (congenital stationary night blindness and multiple congenital ocular anomalies), as well as horse-specific melanoma. The pigmentation phenotype, disorder phenotype, mode of inheritance, genetic or genomic methods utilized to identify the genes involved and, if known, the causative mutations, molecular interactions and other susceptibility loci are discussed. As our understanding of pigmentation in the horse increases, through the use of novel genomic tools, we are likely to unravel yet unknown pleiotropic effects and determine additional interactions between previously discovered loci.     

MicroRNAs与疾病和发育

作为模式生物实验系统,线虫可用于研究控制动物发育和人类疾病遗传机制。研究发育缺陷的线虫突变体有助于在动物中发现对发育和生理过程有重要调控作用的基因。其中一些基因编码一类小RNA,如microRNA(miRNA),通过作用于特定基因信使RNA来调控其蛋白质表达。一些在线虫发育过程中有功能的miRNA在人体中也存在。它们参与调控与疾病相关的生物学过程,如癌症、糖尿病和神经退行性疾病。通过分析miRNA在临床样品、哺乳动物细胞和模式生物线虫中的表达,从而揭示miRNA调控途径在相关人类疾病中的功能。     

Mapping Approaches to Gene Identification in Humans

Although a number of human genes that cause disease have been traced through the defective product, most genetic defects are recognized only by phenotype. When the biochemical defect is unknown, a gene can be located only through molecular approaches based on coinheritance (genetic linkage) of the disease phenotype with a particular allele of a polymorphic DNA marker that has already been mapped to a specific chromosomal region. Linkage studies in affected families have already localized genes for several important diseases, including cystic fibrosis. Finding a genetic linkage in families in which a disease segregates requires that the human genetic map have a large number of polymorphic markers; when the map is dense enough, any disease gene can be located by linkage to a known marker. Many DNA segments with a high degree of polymorphism are being found and mapped as markers in normal reference pedigrees. Genetic linkage mapping has implications even broader than its application to prenatal diagnosis or therapeutic strategy; analyzing mutations in important genes will illuminate basic mechanisms in molecular biology and the early events that lead to cancer and other disorders.     

Genetics of limb anomalies in humans.

The limbs have an essential function in all vertebrates. In animals, the key genes that are involved in the growth and patterning of the limb buds, and of the development of the complex extremities, have been identified and their interactions recognized. Aided by these discoveries, human genetics has also been able to identify, or at least localize, certain genes responsible for anomalies of the limbs. These malformations are isolated or associated with anomalies of other developmental fields, according to the expression domain of the gene involved. Increasing knowledge of the embryology and genes involved has lead to a regrouping of malformation manifestations in genetics terms. Clear genotype-phenotype correlations are difficult to establish owing to the interlinking network of genetic signals underlying limb development.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

The power of zebrafish models for understanding the co‐occurrence of craniofacial and limb disorders

Craniofacial and limb defects are two of the most common congenital anomalies in the general population. Interestingly, these defects are not mutually exclusive. Many patients with craniofacial phenotypes, such as orofacial clefting and craniosynostosis, also present with limb defects, including polydactyly, syndactyly, brachydactyly, or ectrodactyly. The gene regulatory networks governing craniofacial and limb development initially seem distinct from one another, and yet these birth defects frequently occur together. Both developmental processes are highly conserved among vertebrates, and zebrafish have emerged as an advantageous model due to their high fecundity, relative ease of genetic manipulation, and transparency during development. Here we summarize studies that have used zebrafish models to study human syndromes that present with both craniofacial and limb phenotypes. We discuss the highly conserved processes of craniofacial and limb/fin development and describe recent zebrafish studies that have explored the function of genes associated with human syndromes with phenotypes in both structures. We attempt to identify commonalities between the two to help explain why craniofacial and limb anomalies often occur together.     

Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns

ABSTRACT: BACKGROUND: Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Grasbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult. METHODS: We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients. RESULTS: Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved. CONCLUSIONS: Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of Cbl malabsorption. Early diagnosis improves the lifelong care required by these patients and prevents potential neurological long-term complications. This study provides the first comprehensive overview of the genetics that underlies the inherited Cbl malabsorption phenotype.     

Whole Genome Sequencing of Newly Established Pancreatic Cancer Lines Identifies Novel Somatic Mutation (c.2587G>A) in Axon Guidance Receptor Plexin A1 as Enhancer of Proliferation and Invasion

The genetic profile of human pancreatic cancers harbors considerable heterogeneity, which suggests a possible explanation for the pronounced inefficacy of single therapies in this disease. This observation has led to a belief that custom therapies based on individual tumor profiles are necessary to more effectively treat pancreatic cancer. It has recently been discovered that axon guidance genes are affected by somatic structural variants in up to 25% of human pancreatic cancers. Thus far, however, some of these mutations have only been correlated to survival probability and no function has been assigned to these observed axon guidance gene mutations in pancreatic cancer. In this study we established three novel pancreatic cancer cell lines and performed whole genome sequencing to discover novel mutations in axon guidance genes that may contribute to the cancer phenotype of these cells. We discovered, among other novel somatic variants in axon guidance pathway genes, a novel mutation in the PLXNA1 receptor (c.2587G>A) in newly established cell line SB.06 that mediates oncogenic cues of increased invasion and proliferation in SB.06 cells and increased invasion in 293T cells upon stimulation with the receptor’s natural ligand semaphorin 3A compared to wild type PLXNA1 cells. Mutant PLXNA1 signaling was associated with increased Rho-GTPase and p42/p44 MAPK signaling activity and cytoskeletal expansion, but not changes in E-cadherin, vimentin, or metalloproteinase 9 expression levels. Pharmacologic inhibition of the Rho-GTPase family member CDC42 selectively abrogated PLXNA1 c.2587G>A-mediated increased invasion. These findings provide in-vitro confirmation that somatic mutations in axon guidance genes can provide oncogenic gain-of-function signals and may contribute to pancreatic cancer progression.     

The Genetic Basis of Mendelian Phenotypes: Discoveries,Challenges, and Opportunities

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.     

Identifying the genetic basis of ecologically and biotechnologically useful functions of the bacterium Burkholderia vietnamiensis

Signature-tagged mutagenesis (STM) was used to identify genetic determinants of fitness associated with two key ecological processes mediated by bacteria. Burkholderia vietnamiensis strain G4 was used as a model bacterium to investigate: phenol degradation as a model of bioremediation, and pea rhizosphere colonization as a prerequisite to biological control and phytoremediation. A total of 1900 mutants were screened and 196 putative fitness mutants identified; the genetic basis of 137 of these mutations was determined by correlation to the G4 genome. The phenol-STM screen was more successful at identifying phenol degradation mutations (83 mutants; 4.4% hit rate) than a conventional agar-based phenol screen (49 mutants, 5319 screened, 0.92% hit rate). The combination of both screens completely defined the components of the TOM pathway in strain G4 and also identified novel accessory genes not previously implicated in phenol utilization. The rhizosphere-STM screen identified 113 mutants (5.9% hit rate); 107 had reduced tag signals indicative of poor rhizosphere colonization (Rhiz-), while six mutants produced high hybridization signals suggesting increased rhizosphere competence (Rhiz+). Competition assays confirmed that 69% of Rhiz- mutants tested (24/35) were severely compromised in their rhizosphere fitness. Seventy Rhiz- mutations mapped to genes with the following putative functions: amino acid biosynthesis (25; 36%), general metabolism (18; 26%), hypothetical (9; 13%), regulatory genes (4; 5.7%), transport and stress (2 each; 2.8% respectively). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three Rhiz+ mutants inactivated within a single virulence-associated autotransporter adhesin gene; this mutation consistently produced a hyper-colonization phenotype suggesting a highly novel role for this surface adhesin during plant interactions. Our study has shown that STM can be successfully applied to ecologically important microbial interactions, defining the underlying genetic systems important for biotechnological fitness of environmental bacteria such those from the Burkholderia cepacia complex.     

Genetics of rheumatoid arthritis: confronting complexity

The genetic basis for rheumatoid arthritis (RA) is likely to be extremely complex. Even the role of MHC genes remains to be fully defined, and may involve interactive genetic effects. The difficulty of precisely defining the clinical phenotype, as well as underlying genetic heterogeneity, complicates the problem. In addition, stochastic genetic or physiologic events may contribute to the low penetrance of susceptibility genes. This situation parallels developing paradigms for other autoimmune disorders, in which many different genes each appear to contribute a small amount to overall risk for disease, and where severity and specific phenotypic subtypes are subject to genetic effects. The completion of the human genome project, along with advances in informatics, will be required to reach a deeper understanding of RA. It is likely that this will involve an iterative and interactive process between several different scientific disciplines.     

Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.