Application Of Molecular Genetics Research Articles

Establishment of Stably EBV-Transformed Cell Lines from Residual Clinical Blood Samples for Use in Performance Evaluation and Quality Assurance in Molecular Genetic Testing

Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.

How to identify the genetic basis of gastrointestinal and liver diseases?

New insights into the genetic basis of disease are being generated at an ever increasing rate. This explosion of information was ignited by technological advances, such as the polymerase chain...

Poxviruses as vaccine vectors

The discovery of Jenner in 1798 founded the science of immunology and eventually led to smallpox eradication from the earth in 1980 after a world-wide vaccination campaign with vaccinia virus (another poxvirus) and paradoxically, despite the eradication of smallpox, there has been an explosion of interest in vaccinia virus in the eighties. This interest has stemmed in part from the application of molecular genetics to clone and express foreign genes from recombinant vaccinia viruses. Vaccinia is also gaining renewed interest due to bioterrorism. These recombinant viruses have multiple applications in research and vaccinology and led to the development of vectored vaccines, such as the recombinant vaccinia rabies vaccine used to eliminate rabies in Western Europe and, more recently, in the United States. Secondly, alternative poxvirus vectors, such as avipox viruses, were proved to be even safer and efficacious non-replicating vectors (suiciole vectors) when used in non-avian species.

Linkage analysis in schizophrenia using a neurocognitive phenotype

Linkage disequilibrium (LD), the observed correlation between alleles at different loci in the genome, is a determinant parameter in many applications of molecular genetics. With the wider use of genomic technologies in animal breeding and animal genetics, it is worthwhile revising and improving the current knowledge and understanding of cattle LD.This study analyzes levels of LD assessed through the r2 measurement in seven breeds of cattle from both indicine (Bos indicus) and taurine (Bos taurus) (sub)species, genotyped with a high density panel (HD) of over 777,000 single nucleotide polymorphism (SNP) markers. Average levels of LD were assessed for inter-marker distances from 0 kb up to 10 Mb. Two more SNP panels were built in silico from the original genotypes to represent a lower density SNP chip including approximately 50,000 SNPs (50K) and a panel of 249,000 SNPs chosen to comply with specific minor allele frequency (MAF) distributions.The results show higher levels of LD than previously reported, especially at short inter-marker distances of less than 20 kb, assessed through the use of the higher density panel. Negligible impact of MAF changes in the estimation of r2 was observed, supporting the use of the HD panel for LD-based methodologies even when ascertainment bias in the choice of SNPs is suspected. The 50K SNP panel indicated lower levels of LD in the first 100 kb and highly unreliable LD estimates for distances shorter than 50 kb. Overall, lower levels of LD were observed for all indicine breeds as compared to taurine breeds, with larger differences assessed in the first 100 kb, suggesting the need for use of higher SNP densities in this (sub)species when applying LD-dependent methods, as compared to most taurine breeds.

The molecular architecture of the inner ear.

The inner ear is structurally complex. A molecular description of its architecture is now emerging from the use of contemporary methods of cell and molecular biology, and from studies of ontogenetic development. With the application of clinical and molecular genetics, it has now become possible to identify genes associated with inherited, non-syndromic deafness and balance dysfunction in humans and in mice. This work is providing new insights into how the tissues of the inner ear are built to perform their tasks, and into the pathogenesis of a range of inner ear disorders.

Changes in age‐associated neurodegenerative diseases in the last half century

Age‐associated neurodegenerative diseases are a serious and growing problem for mankind, and their etiologies are still unknown. The author has been studying the neuropathology of fatal neurodegenerative diseases endemic to the native Chamorro population on Guam during the last half a century. During this time, remarkable advances in scientific technology, such as the application of electron microscopy, immunohistochemistry and molecular genetics, have contributed to numerous discoveries in classic neurological diseases. In addition, various new diseases have been recognized. This communication reviews Alzheimer's neurofibrillary tangles and Cu/Zn superoxide dismutase positive Lewy body‐like inclusions in the anterior horn cells, in order to elucidate these pathological findings as well as remarkable changes observed in certain other age‐related neurodegenerative diseases.

Entendendo o Processo Molecular da Tumorigênese

Nos últimos 25 anos, o reconhecimento dos mecanismos genético-moleculares implicados na gênese e na progressão do câncer tem permitido obter novos métodos de diagnóstico e de acompanhamento, redirecionando de forma drástica a terapêutica do paciente com neoplasia. Alguns marcadores moleculares já estão sendo utilizados na rotina e deverão prover testes sensíveis e específicos para o diagnóstico precoce, estadiamento e acompanhamento do paciente com câncer. As características moleculares de cada tumor deverão permitir predição do seu comportamento, ajudando a delinear estratégias terapêuticas mais efetivas. Apresentamos de forma didática os principais mecanismos controladores do ciclo celular e do crescimento, definindo a importância de oncogenes erroneamente ativados e de genes supressores tumorais perdidos ou não-funcionantes, dos genes envolvidos na programação e manutenção da vida celular e de outros genes que atuam no processo de tumorigênese. Os mecanismos de progressão tumoral, invasão e metastatização à distância são revistos enfatizando-se a aplicação prática do conhecimento a respeito de tais mecanismos. Lembramos o papel da instabilidade genética e dos fenômenos epigenéticos na definição fenotípica do câncer, sugerindo as aplicações da genética molecular na terapia gênica do câncer.

Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data.

Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.

Genetics of ventricular tachycardia.

Pathogenesis of familial inherited arrhythmias is being progressively clarified thanks to the insights provided by molecular biology and by functional studies. Transmembrane or intracellular ion channel mutations have been identified in genetically determined forms of polymorphic ventricular tachycardia and sudden death such as catecholaminergic ventricular tachycardia, long QT syndrome, and Brugada syndrome. The role of molecular abnormalities in the genesis of monomorphic idiopathic ventricular tachycardias is less well defined, mainly because of the lack of a Mendelian pattern of inheritance. Interestingly, the presence of somatic mutations has been suggested as the mechanism for monomorphic ventricular tachycardia originating from the right ventricular outflow tract. The future goals for the application of molecular genetics to the management of cardiac arrhythmias will be to apply molecular genetics for a better risk stratification of affected individuals and to aim for the identification of gene-specific treatment of idiopathic ventricular tachycardia.

Applications of molecular genetics to the understanding of congenital ocular motility disorders.

The congenital fibrosis syndromes (CFS), including congenital fibrosis of the extraocular muscles (CFEOM) and Duane syndrome (DS), are rare congenital strabismus syndromes that present with nonprogressive restrictive ophthalmoplegia with or without ptosis. Although historically believed to result from primary extraocular muscle (EOM) fibrosis, our laboratory's work is based on the hypothesis that these disorders result from distinct, but analogous, developmental defects of the oculomotor (nIII), trochlear (nIV), and abducens (nVI) nuclei. We have defined three inherited CFEOM phenotypes (CFEOM1-3) and have mapped each phenotype to a distinct genetic locus (FEOM1-3). Individuals with CFEOM1 are born with bilateral ptosis and both eyes fixed in a downward position with absent upgaze and aberrant horizontal gaze. This disorder maps to the FEOM1 locus on chromosome 12cen.(1,2) Neuropathology studies of CFEOM1 reveal the absence of the superior division of oculomotor nerve and its corresponding alpha motor neurons in the midbrain, with abnormalities of target EOMs.(3) These neuropathology findings parallel those previously identified in Duane syndrome, in which there is an absence of nVI and the abducens nerve.(4,5) Individuals with CFEOM2 are born with bilateral ptosis and exotropia. This atypical form of CFEOM maps to the FEOM2 locus on chromosome 11q13 and results from mutations in ARIX (PHOX2A).(6,7) ARIX encodes a homeodomain transcription factor protein previously shown to be required for nIII/nIV development in mouse and zebrafish.(8,9) Together, these findings support the hypothesis that the congenital fibrosis syndromes result from parallel defects in nIII, nIV, and nVI nuclear development. Functional studies of the CFEOM genes should provide additional insight into the unique features of the extraocular lower motor neuron axis in health and disease. (For full (refs. 1-9), see reference list of the main paper.)

Alzheimer's disease: molecular understanding predicts amyloid-based therapeutics.

Degenerative diseases of the brain were long considered among the most obscure and intractable of human maladies. However, recent advances in understanding their mechanisms have brought us to the verge of potential disease-modifying agents. This progress is perhaps best exemplified by the case of Alzheimer's disease. The application of molecular pathology and genetics has led to the recognition that the four genes implicated to date in familial Alzheimer's disease all chronically elevate cerebral levels of the amyloid beta-protein (Abeta). Accordingly, small molecule inhibitors of the beta- and gamma-secretases, the proteases that generate Abeta from its precursor, are under active development, and some have shown in vivo efficacy in mouse models. An alternative approach, active or passive immunization against Abeta, has received extensive pre-clinical validation in mice, but an effective preparation free of significant side effects in humans is still awaited. Several other potential therapies are also reviewed here. If one or more of these varied approaches is ultimately proven to slow or prevent dementia, Alzheimer's disease will become a salient example of the successful application of reductionist biology to the most complex of organs, the human cerebral cortex.

British Society of Human Genetics

British Society of Human Genetics

Taxol: biosynthesis, molecular genetics, and biotechnological applications.

Over the past decade, Taxol and its closely related structural analogue Taxotere have emerged as very important antitumor agents. Their widespread use in the treatment of a variety of cancer types, their likely approval for the treatment of additional forms of cancer, and their use at earlier stages of intervention will lead to increased demand for these drugs in the future. Because of yield considerations, Taxol and Taxotere are currently derived via semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which must be isolated from yew (Taxus) trees. Thus, efforts are underway to produce Taxol (and other advanced taxoids for use in semisynthesis) by alternate, biotechnological means. This article provides a current overview of research on taxoid biosynthesis and an assessment of bioengineering applications for taxoid production in yew cell culture.

Clinical Oncology

2nd ed, edited by Martin D. Abeloff, James O. Armitage, Allen S. Lichter, and John E. Niederhuber, 2963 pp, with illus, Philadelphia, Pa, Churchill Livingstone, 2000.The second edition of Clinical Oncology is a comprehensive overview of human cancer as it stood in the year 2000. The editors are to be commended for gathering together and publishing a wide array of thorough yet very readable reviews on a wide spectrum of topics. As described in the “Preface” to the second edition, the intent of the editors was to provide a substantial knowledge base in an accessible format that would be useful to a wide range of individuals. The target audience includes practicing oncologists, primary care physicians, and nononcologic specialists, as well as students, residents, and fellows undergoing active study and training. This goal has been successfully achieved.The textbook is laid out in 4 parts. Each part is separated into various sections, totaling 96 chapters, and each includes thorough discussions of state-of-the-art technology and therapeutic approaches. Part I of the textbook is organized into 5 sections: “Genetic Basis of Cancer,” “Immunologic Basis of Cancer,” “Environmental Basis of Cancer,” “Principles of Prevention, Diagnosis, and Treatment,” and “Understanding Risk.” Part I thus provides the reader with a thorough scientific overview of modern oncology. There is a strong emphasis on the implications and applications of molecular genetics in the practice of oncology. Based on our ever-increasing understanding of the immune system and biologic processes, such as angiogenesis and signal transduction, part I also focuses on new developmental therapies arising from this understanding.Parts II, III, and IV of Clinical Oncology focus on problems common to cancer and its therapy, management of specific malignancies, and rehabilitation of the cancer patient, respectively. The 6 sections of part II, entitled “General Problems,” “Hematologic Problems,” “Infections,” “Metabolic Problems,” “Surgical Problems,” and “Local Effects of Cancer and Its Metastasis,” are familiar areas to all those who deal with and treat cancer patients. These discussions are well organized and referenced, so much so that this part of the textbook could easily stand alone in its own right. The authors of all 42 of the chapters in part III, “Specific Malignancies,” have done an outstanding job of not just cataloguing recent information, but also emphasizing their own interpretation of data. Each of these chapters or chapter sections includes a quick orientation of the subject matter in summary form at its opening, allowing the reader an overview of the discussion. A unique and valuable aspect of these patient management chapters is the display of personal strategies and algorithmic approaches to clinical problems in nonreferenced editorial sections. This information is set off from the main text and highlighted in red so that the reader can easily identify these personalized approaches. The 3 chapters in part IV, “Rehabilitation of the Cancer Patient,” are valuable and essential components to the comprehensive care of these patients. These chapters, entitled “Physical Rehabilitation,” “Evaluation and Management of Sexual Dysfunction,” and “Psychosocial Rehabilitation,” once again give a personalized overview of the rehabilitative approach.This edition of Clinical Oncology brings together a balanced overview of all aspects of cancer medicine, including the scientific, clinical, and humanistic disciplines, which affords the reader an invaluable resource. It is highly recommended.

The genetic causes of neurodegenerative diseases.

With the application of molecular genetics, we are now beginning to understand the etiology and the early stages of pathogenesis of the major neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Pick's disease and Progressive Supranuclear Palsy. Surprisingly, these studies are showing that these diseases share pathogenic mechanisms which involve tau or synuclein aggregation. In this article, I review the progress in the molecular genetic analysis of these major neurodegenerative diseases and discuss how they are related to each other.

Design and implementation of an introductory course for computer applications in molecular genetics. A case study.

Formal training in computational biology was initiated at Wayne State University in 1990 to meet the needs of the faculty. This was still at a time when the molecular databases and analysis tools could be housed in what is now equivalent to a modern but dated desktop computer. In 1995 the course was expanded to include graduate students to provide these senior students with a foundation in computational biology. This course has armed our students with a requisite set of basic skills that are necessary for a successful career in molecular genetics. It is now an integral component of the graduate program of the Center for Molecular Medicine and Genetics and our experiences in course delivery have been detailed (BioInformatics Methods and Protocols, S. Misener and S. A. Krawetz, eds., Humana Press, Totowa, NJ, 2000.). The course was expanded to a campus-wide unlimited enrollment program for the summer of 2000 to address the needs of our student body. In this review we present our experience with delivering a multidisciplinary campus-wide computational biology course to a new and widely diverse student body.

Haemochromatosis: novel gene discovery and the molecular pathophysiology of iron metabolism.

The application of molecular genetics to haemochromatosis and experimental mutagenesis in animals has transformed our capacity to investigate the unique physiology of iron homeostasis-a key problem in biology and medicine. The identification of HFE, the principal determinant of adult haemochromatosis (HFE1; OMIM 235200) and TfR2, recently implicated in a rarer form of the inherited disorder (HFE3; OMIM 604250), and the promise of candidate genes for juvenile haemochromatosis (HFE2; OMIM 602390) and neonatal haemochromatosis (OMIM 231100) provide the foundation for important studies into the control mechanism of iron balance in humans. The rare conditions atransferrinaemia (OMIM 209300) and acaeruloplasminaemia (OMIM 604290), each associated with tissue iron overload, have already implicated the iron transport ligand transferrin and the copper transporter caeruloplasmin in the control of iron homeostasis. Gene mapping studies in animal mutants with anaemia due to defects in the uptake or tissue transfer of iron have yielded novel proteins involved in iron transport: DMT1 (brush border transporter of ferrous iron) in the mk/mk mouse, hephaestin (basolateral multi-copper ferroxidase) in the sex-linked anaemic mouse (sla) and ferroportin1 (basolateral iron exporter) in zebrafish weh mutants. The discovery of genes that determine heritable defects of iron absorption and regulation in animals and humans thus holds promise for a complete mechanistic understanding of the molecular pathophysiology of iron metabolism.

Implications of genetic epidemiology for the prevention of substance use disorders.

Despite advances in characterizing human genotypes, the complex process through which genes exert their influence limits the application of molecular genetics to human diseases. Substance use disorders are necessarily complicated by gene-environment interaction because exposure to an exogenous substance is required for their development. The methods of genetic epidemiology are specifically designed to identify sources of complexity that impede etiologic findings and prevention efforts. The goal of this paper is to illustrate the application of family study methods to identify risk factors for substance abuse and their implications for prevention. The Yale Family Study is a controlled family study of the comorbidity of substance and psychiatric disorders. The sample consists of 223 probands with substance use and/or an anxiety disorders and community controls, 1218 adult first degree relatives and spouses, and 203 offspring (ages 7-17) followed for 8 years. Results indicated familial aggregation of substance disorders in adults and children, independence of familial aggregation of alcoholism and drug dependence, and specificity of familial clustering of some drugs of abuse. Familial factors are more strongly associated with substance dependence than abuse, with an attributable risk of 55%. Premorbid psychiatric disorders--social phobia and bipolar affective disorder in adults, and depression, anxiety, conduct, and oppositional defiant disorders in children--were strongly associated with the subsequent development of substance dependence (attributable risks ranging from 44 to 86%). A family history of substance abuse and premorbid psychopathology are strongly associated with the development of substance use disorders. Implications for primary and secondary prevention are discussed. As specific genetic vulnerability markers for substance use disorders become identified, application of the tools of genetic epidemiology may be employed to identify specific environmental risk factors that may serve as targets for prevention.

ComboScreen facilitates the multiplex hybridization-based screening of high-density clone arrays.

The construction of physical maps based on bacterial clones [e.g. bacterial artificial chromosomes (BACs)] is valuable for a number of molecular genetics applications, including the high-resolution mapping of genomic regions of interest and the identification of clones suitable for systematic sequencing. A common approach for large-scale screening of bacterial clone libraries involves the hybridization of high-density arrays of immobilized, lysed colonies with collections of DNA probes. The use of a multiplex hybridization screening strategy, whereby pooled probes are analysed en masse, simplifies the effort by reducing the total number of parallel experiments required. However, this approach generates large amounts of hybridization-based data that must be carefully analysed, assimilated, and disambiguated in a careful but efficient manner. To facilitate the screening of high-density clone arrays by a multiplex hybridization approach, we have written a program called ComboScreen. This program provides an organizational framework and analytical tools required for the high-throughput hybridization screening of clone arrays with pools of probes. We have used this program extensively for constructing mouse sequence-ready BAC contig maps.

Transactivation of a target gene through feedforward loop activation in plants.

The expression of many genes encoding transcriptional activators in prokaryotes and eukaryotes is upregulated through positive feedback activation. During positive feedback activation, a transcriptional activator binds to its own promoter and thus increases its own expression as well as the expression of its target genes. In the simplest case, increased levels of the transcriptional activator can be directly correlated with increased expression of its target genes. In this study, we present a gene expression system, designated feedforward loop (FFL) system, which makes use of this kind of positive feedback regulation for the expression of plant transgenes. We show in transient and stable transformation experiments that such a system is functional and that it can be used to obtain high level gene expression in plants. We also provide evidence that the transgene is ubiquitously expressed when using the FFL system. Finally, we discuss the possibilities of using FFL gene expression systems for applications in plant molecular genetics and biotechnology.