Opportunities and Limitations of DNA Screening Technologies - Essay Example

Limitations of DNA Screening Technologies Introduction Genetic testing for the purpose of identifying disease genes has become one of the most widely used tools in molecular medicine. The search for the genetic origins of inherited disease syndromes has led to the identification of several thousand genes responsible for many single gene disorders (Cotton, 1993). The molecular technologies that have facilitated this search have also led to the assessment of specific mutations and their effects on protein structure and function that have been critical to the development of an understanding of physiological mechanisms of pathogenesis (Balogh, 2004). The plethora of genetic testing methodologies, however, has not led to the expected breakthroughs in treatment and prevention of diseases with a genetic component, as anticipated. This paper will review the important technologies as applied to the study of genetic disorders, their strengths and limitations in clinical genetics and molecular medicine (Cotton, 1993).   Genetic screening methodologies used in detection of human disease genes The basic tools used in molecular screening of inherited genetic disorders were developed in the 1980s and 1990s and led to the identification of several hundred genes involved in the genesis of human single gene disorders (Altschuler, 2008). These discoveries were enhanced by the results of the DNA sequence analysis of the human genome in the early days of this century. Today, clinicians and researchers alike are afforded a close-up molecular view of the human genome, which has opened the door to molecular assessments of gene structure and function that were unthinkable a half century ago (Orrita et al, 1989). The tools of molecular medicine are largely built upon the intrinsic chemical and biophysical properties of DNA (Cotton, 1993). Among the most important of these in terms of their methodological applications, is the base pair complementarity of the nucleotide bases which allows single stranded DNAs of specific nucleotide sequences to function as probes for molecular hybridization to DNA targets with similar complementary base sequences (Altschuler, 2008). The application of this chemical property of DNA is the basis of blot hybridization methods of Southern blot analysis, DNA fingerprinting, genomic in situ hybridization, Northern blot analysis, DNA microarrays, and others (Keen et al, 1991). The tools of DNA sequence analysis are comprised of the dideoxy method developed by Frederick Sanger (Sanger et al, 1977). This important methodology is a direct application of the physiological mechanism of semi-conservative DNA replication. the identification of DNA base sequence. The application of this technology and the use supercomputers to speed up the analysis of sequence data was the technological basis of the human genome project that accomplished the identification of all three billion bases of human DNA within several years (Wine et al, Another basic molecular tool based on the fundamental properties of DNA is gel electrophoresis (Meyers et al, 1985). This methodology employs the use of porous gels and electric current to effect the separation of linear DNA molecules based on their physical properties. These properties include the uniformly distributed negative charge provided by the alternately placed phosphate groups of the sugar phosphate backbone and the fixed diameter of the double helix, such that DNAs of different molecular weights are comprised of difference in the linear number of base pairs. This simple biophysical parameter can be used to identify the molecular weight of DNA as the distance migrated by a linear DNA through a porous DNA of agarose or polyacrylamide is inversely proportional to the log of the molecular weight of the linear molecule measured in number of base pairs (bp) (Wilson et al, 1987). Gel electrophoresis is a standard tool of DNA molecular weight determination as it relates to length differences used in gene mapping and fingerprint analysis and single base length differences that comprise the basis of DNA sequence analysis (Myers et al, 1985). Applications of DNA technology to the study of inherited genetic disease The plethora of data that has accumulated over the past decade on genes associated with physiological dysfunction has generated a monumental task for assessment (Altschuler, 2008). Even in the context of genetic disorders that result from defects in only a single gene, many hundreds of mutations have identified in some cases (Forrest et al, 1995). In contrast to sickle cell disease, which is the result of only one major and several total mutations in the beta globin gene (Wilson et al, 1997), there are other disorders, such as cystic fibrosis, in which over 800 different mutations have been identified in the CF gene in patients suffering from this serious lung disorder (Carlsson et al, 1996; Wine et al, 2001). There are a number of testing tools that have been developed to screen patients with CF to detect the precise mutation present in an individual case; this may involve multiple screenings using a battery of testing tools that have been developed by biotech companies for this purpose (Wine et al, 2001). The end result is that after a long and expensive testing process, the patient with CF will be given a base sequence analysis of the specific mutation in the CF gene responsible for his/her disease (Kerem et al, 1989). This is only one example of the many screening tests that are frequently used to identify the molecular mutation responsible for a given disease when the single gene disorder may be the result of numerous mutations within a single gene. Other examples include phenylketonuria and the thalassemias (Carlsson et al, 1996). When these diagnostic tools were first used in the assessment of the molecular basis of gene dysfunction, geneticists were astonished to find that so many genes involved in the genesis of single gene disorders contained many different types of mutation in different parts of these genes, any of which could be the primary cause of the phenotypic effects on protein function and associated pathophysiology (Altschuler, 2008). These studies were extremely important as they provided valuable evidence of the molecular basis of disease, including the mapping of functional domains within a gene and its cognate protein and associating specific molecular consequences to subtle changes in the structure and function of proteins resulting from individual mutations (Forrest et al, 1995). In addition, these assessments were invaluable to population studies on the aetiology of single gene disorders, mutational hotspots within the genome and the epidemiological assessment of origins and persistence of genetic disorders in different human populations (Carlsson et al, 1996). Limitations of genetic screening methodologies Clinical diagnostic catalogues of the molecular mutations associated with different single gene disorders have been incorporated into enormous databases such as OMIM that serve as a repository of mutational assessment data (Carlsson et al, 1996). For all except the rarest of single gen disorders, a complete catalogue of mutations has been generated from ongoing patient screening over the past few decades (Altschuler, 2008). From a clinical standpoint, the enormous spectrum of mutations associated with a given gene for the most part, has been shown to produce minor, if any, differences in the severity or symptomatology for most single gene disorders (Altschuler, 2008). There are certain phenotypic manifestations of disease that may correlate with certain mutations; for example, certain mutation profiles of cystic fibrosis correlate with associated pancreatic insufficiency while others do not (Kerem et al, 1989). Beyond these broad categories, most mutational differences within a single gene do not translate to difference in disease phenotype associated with clinical severity, prognosis of therapeutic indications (Cotton, 1993). In this context, it is relevant to question the widespread application of these available, but expensive and time consuming screening tests to all patients diagnosed with a specific single gene disorder (Lau & Leung, 2005). While it may be highly appropriate to use general genetic screening tests in prenatal screening for the detection of genetic disease, the involved genetic assessment of the molecular basis of mutation in individuals already diagnosed with a specific single gene disorder may be unnecessary and unproductive with respect to clinical impact (Meyer, 1995). Unfortunately, the development of technological applications with ever-increasing specificity and sophistication has sometimes led to the notion that, once in existence, the technology must be utilized, without regard to its overall contributory value to clinical assessment, management or even prevention of single gene disorders (Meyer, 1995). This criticism is not simply economically driven, but also reflects questions of the judicious application of technological resources to the management of genetic disease. What makes this discussion most relevant is the observation that, despite the widespread implementation of the tools of biotechnology to the molecular assessment of genetic disease, in very few instances have these studies led to any new developments in the treatment or clinical management of single gene disorders over the past several decades (Lau & Leung, 2005). One might inquire as to whether better use might be made of the economic resources and research strategies in order to approach these disorders in ways that might produce greater clinical benefit to patients suffering from genetic diseases. The ultimate goal of clinical genetics research in this area is to develop gene therapy approaches that may intervene at the level of correcting the dysfunctional gene by gene replacement, termed corrective gene therapy (Carlsson et al, 1996). This is an important area of research; however, there is no way to predict how long it will take to make progress in this area (McQueen, 2002). In the interim, it is important that geneticists and clinical researchers approach each single gene disorder in the multi-factorial context in which it exerts its physiological manifestations (Meyer, 1995). For the most part, many of the physiological consequences of single gene disorders result not only from the proscribed effects of a single mutation on the function of a specific protein, but also reflect the broad spectrum physiological consequences of the initial dysfunction that often includes environmental factors and pathophysiological responses that affect disease severity and prognosis (Altschuler, 2008). A very simple example involves phenylketonuria (PKU), a single gene disorder that results from the absence of the enzyme responsible for metabolising the amino acid phenylalanine, phenylalanine hydroxylase (McQueen, 2002). The accumulation of phenylalanine and its byproducts results in severe damage to the developing brain. To prevent this catastrophic brain damage, infants diagnosed with PKU are given a diet containing very low levels of phenylalanine, to prevent brain damage (Schuber et al, 1997). As a result, most children with PKU develop normally without any central nervous system deficit. Not all genetic diseases, unfortunately, can be approached in this way. However, the study of multi-factorial components of genetic disease may lead to better treatments and patient outcomes (Schuber et al, 1997). For example, the progressive destruction of the lungs of patients with cystic fibrosis is not the direct effect of the CF mutation on the chloride transmembrane channel conductance, but rather due to the fact that these patients invariably develop infections with the antibiotic resistant microbe Pseudomonas aeruginosa, that ultimately destroys the lungs (McQueen, 2002). Perhaps a better use of resources currently focused on the identification of which of the 800 mutations of the CF gene is present in each newly diagnosed patient, would be to utilise research efforts to develop antibiotics to treat infections caused by P.aeruginosa. Conclusion The development of molecular tools for genetic screening has created a revolution in our understanding of the molecular basis of genetic disease, facilitating the discovery of disease-causing genes as well as the molecular mechanisms of pathogenesis. These technologies also offer promise for the development of gene therapy approaches that may someday lead to the eradication of these terrible diseases. The unrestricted application of these technologies to patient assessment, however, has been, for the most part, unproductive in the development of new therapeutic approaches to the treatment of single gene disorders. It is important to assess the entire clinical picture rather than simply focusing on its genetic components to develop new approaches to the clinical management of genetic disease. Word count 1972 Reference List Altshuler D, et al 2008, Genetic mapping in human disease. Science (New York, N.Y.) vol. 322, no. 5903, pp. 881-888. 169. 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