Current Molecular Techniques in Genetic Disease Diagnostics - Coursework Example

Current Molecular Techniques in Genetic Disease Diagnostics Introduction The discovery of the main principles behind genetics, heredity, and DNA in the twentieth century has led to many advances in the field of science, specifically medicine. The important personages in the century are Gregor Mendel (the ‘Father of Genetics”), Avery (DNA as the genetic material; Avery, McLeod and McCarty 1944); Watson and Crick ((Watson and Crick 1953; the double helix structure of the DNA which is made up of only four nucleotides) and Nirenberg (genetic code is made up of simple three letter codes). The human genome is made up of approximately 30,000 genes. In each gene, there are exons and introns which are involved in forming mature messenger RNA. Messenger RNA or mRNA is translated into protein. Through different combinations and cutting of exons, genes may produce different mRNA and consequently different proteins. Thus, there are more proteins than genes in our cells. Significant changes, like deletion or insertion of nucleotides (called mutations), in gene structure can lead to the production of abnormal proteins that may result in disease. Hereditary diseases are occur when the changes in gene sequence is passed on to offspring. Polymorphism in coding regions of genes produce what are known as alleles and resulting changes in protein structure can increase the risk for diseases like hypertension and schizophrenia. Mutations which occur only in body cells (somatic cells) include cancer. Development of Genetic Diseases The human body recognizes the significance of DNA as the genetic material or the blueprint of life. Thus, a complex system of proteins provides support to the DNA replication process, making sure that genomic stability is maintained at all cost. Due this system, DNA can be copied with high fidelity for billions of times. Few mutations occur during this process because the mistakes in copying and damage to DNA are sensed by several proteins which oversee the repair of the damage or death of the cells with the damaged DNA. When the genes for these guardian proteins become mutated, then genetic stability is destroyed. Inherited mutations predispose the cells to develop cancer and other irregularities in cell division. Genetic instability in cancer cells result in weaker cells with increased susceptibility to damage and further mutations which result in higher malignancy and resistance to treatment. Genetic disease also results from defects in the regulation of gene expression. Disease occurs when there is a mutation in the gene for a certain transcription factor. Mutations in signalling molecules affect the cell’s response to environmental factors and expression of downstream genes. This can lead to cell proliferation and accumulation of disease-causing genetic factors. Molecular Diagnostics Advances in genome and genetics researches have ushered in a new era of medical diagnostics and disease therapy. Genetic testing is composed of DNA tests to determine the deficiency, excess and abnormalities in the DNA sequence of the diseased organism (in this case, humans) and in pathology, of the disease causing organism. The premier and basic methods which served as the foundation for disease diagnostics are polymerase chain reaction or PCR, which won a Nobel Prize in Chemistry for its discoverer and DNA sequencing. Polymerase chain reaction was invented by Kary Mullis in 1983. With this process, millions of copies of a single strand of DNA can be made in a few hours. With PCR, primers are synthesized for the specific segment that is to be copied. DNA polymerase, DNA building blocks called dNTPS, and buffers complete the mix which is placed inside the PCR machine (Figure 1). After the reaction, copies of the DNA can be isolated using several techniques foremost is through agarose gel electrophoresis followed by purification from the gel. The purified DNA can then be sequenced. The resulting sequence is compared to the original gene sequence for the normal protein to see if there are significant differences which could mean abnormality or disease. Different genes expressed can be isolated by PCR of cDNA produced by mRNA by reverse transcription. Sequence specificity of the primers is emphasized (Liang & Pardee, 1992). Variations and advanced application in PCR technology has led to the development of real-time PCR machines that can monitor and quantify the progression of disease genes with time or in different body tissues. With multiplex PCR, several genes can be mixed and allowed to amplify at a single time (Henegariu, et al. 1997). The DNA sequence data will give the amount of mutations and other changes which can indicate mutations. The Human Genome Project has already identified the normal gene sequences that when altered by even a single nucleotide can result in disease. Among these diseases, the most popular is sickle-cell anaemia an autosomal recessive disease caused by a single nucleotide or point mutation in the haemoglobin beta gene (HBB) which can found on chromosome 11. The mutation results in changing the amino acid from valine in the Beta chain of haemoglobin to glutamine. Under low oxygen levels, the red blood cells with the changed amino acids takes in a sickle cell shape as opposed to the normal round cell shape. The gold standard for scanning mutation is Sanger DNA sequencing (Figure 2), but it is relatively laborious and expensive. These make it difficult to scan for many genes in the genomic region of interest. To address the limitations of the Sanger method, other DNA sequence scanning methods were developed grouped according to presence of heteroduplex mismatches, physical differences of single-stranded DNA and simplified DNA sequencing approaches. Newer methods like surface plasmon resonance (Figure 3) bind GG mismatches using an intercalating ligand bound to a sensor surface. The reflective index of polarized light as affected by the surface bound DNA molecules indicates the degree of GG mismatches in the DNA. Another common diagnostic tool is nucleic acid hybridization with the use of gene fragments from the patient with known complementary genetic and molecular probes. Gene expression is monitored using microarray technology with the use of DNA “chips”, slides on which are embedded thousands of partial gene sequences (for review see Dykes, 1996). These are allowed to hybridize with the complementary DNA sequences from messenger RNA of the patient. A schematic diagram for the general protocol is given in Figure 4. Understanding the molecular mechanism of disease has been possible due to automated and rapid sequencing of DNA. Molecular imaging of cells has also contributed greatly to disease diagnostics. Molecular imaging is built on the traditional techniques such as magnetic resonance, computed tomography, and ultrasound. These all rely on absorption and light scattering characteristics to detect disease. A shift to specific molecular sources of diseases defines molecular imaging techniques from the traditional techniques (reviewed by Weissleder 1999). With this technique, earlier detection in vivo is possible. Advantages of molecular imaging techniques include the evaluation of specific disease parameters (example growht of tumors), effectiveness of therapy used, pathogenesis and rapid three-dimensional information which is not possible with DNA technologies. The imaging techniques used are nuclear imaging, magnetic resonance, optical techniques and positron imaging. Genetic Testing in the United Kingdom The use of genetic testing has become routine for many genes that serve as molecular markers for disease. The United Kingdom Genetic Testing Network (UKGTN) is an umbrella organization of laboratories, hospitals, and medical facilities that offer genetic testing. Currently, there are dozens of diseases that can be identified with genetic testing (for a sample list, see Appendix Table 1) (Molecular Genetics Laboratory 2008). It aims to ascertain that high quality genetic testing services are available to all patients of the National Health System (NHS). New tests are always being evaluated; results of these are used as basis for the recommendation for new services. UKGTN serves as a bridge between clinical diagnostic laboratories that provide genetic testing services for patients and their families who have genetic disorder. Laboratories under UKGTN’s wings have high quality standards and the tests they offer first undergo a rigorous process of evaluation to ensure their scientific validity and clinical utility. Common diseases that are diagnosed by genetic tests are cystic fibrosis, muscular dystrophy, cancer susceptibility fragile X syndrome familial hypercholesterolemia. Most commonly, the tests utilize the molecular techniques of PCR and mutation screening utilizing specific gene primers. The genetic tests that are being carried out can be categorized into several groups (Human Genome Project 2008). Carrier identification refers to the genetic tests by couples with family histories of recessive genetic disorders that could be passed on to their children once they decide to have some. The most common tests are for sickle-cell anaemia, cystic fibrosis, and Tay-Sachs disease. Genetic testing of the fetus is performed with a prenatal diagnosis when the risk of bearing a child with genes associated with mental retardation or other physical defects. The most common genetic disease tested is Down Syndrome. Babies are screened for diseases that are treatable in newborn screening for phenylketonuria and congenital hypothyroidism. Phenylketonuria is an inherited disease due to the deficiency in the enzyme phenylalanine hydroxylase. Without the enzyme, phenylalanine, an amino acid builds up in the body to toxic levels resulting in organ damage and mental retardation. The disease is an autosomal disorder due to mutations in the genes for phenylalanine hydroxylase. Diet can reduce the effects of the disease. All the 52 states in the United States of America conduct newborn screening. Tandem mass spectrometry, a new technology is used to detect additional genetic disorders in newborns in some states in the United States (Khoury, McCabe and McCabe 2003). Tandem mass spectrometry can identify newborns having medium-chain acyl–coenzyme A (CoA) dehydrogenase which results in hypoglycemia, seizures, and death in about 20% of the babies after the initial episode for the first two years of life. Screening for late-onset disorders are for complex adult diseases that result from genetic and environmental causes. Such diseases are heart disease and cancer. The late-onset genetic screening will indicate predisposition to these diseases. Gene expression profiling is one modern tool used to detect predisposition to these diseases (Guttmacher and Collins 2003). Huntington's disease, caused by a single gene HD, is seen later in life but can be tested at any time. The mutation is an expansion of the nucleotide triplet repeat CAG where increased number of triplets lead to decrease in the age when the disease sets it. Huntington’s disease is neurodegenerative leading to dementia. What the Future Holds DNA fingerprints or an individual’s genetic profile can be collected from DNA test results to identify genetic markers that make an individual unique from others. Information from genetic testing is used in legal cases to identify or claim parentage, identify bodies and others. DNA fingerprints used to identify an individual’s potential for negative traits, deadly and contagious diseases still face resistance due to issues in privacy. In the future, the genetic sequence of an individual’s genome will be used to screen potential to succumb or to contract diseases like cancer, hypertension, diabetes, and heart disease. This has advantages because knowing potential risks could result in early intervention and shift to a healthier lifestyle. The availability of genetic testing should be an option for all who think that they are at high risk for developing disease later in life. We should all take advantage of the new technologies towards the betterment of our health and ultimately, our lives. References Avery, Oswald T, Colin M McLeod, and Maclyn McCarty (1944) ‘Studies on the chemical nature of the substance inducing transformation of pneumococcal types’. Journal of Experimental Medicine, Vol 79, no. 2 pp.137-158. Dykes, Colin W. (1996) ‘Genes, disease and medicine’. British Journal of Clinical Pharmacology Vol.42, pp. 683-695. ‘Genes and Diseases.’ (2008) NCBI. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=gnd.TOC&depth=2 (accessed June 12, 2008). Guttmacher, Alan E, and Francis S. Collins. (2003) ‘Molecular diagnosis of the hematologic cancers.’ New England Journal of Medicine, Vol. 348, no. 18 pp. 1777-1785. Henegariu, O, N.A Heerema, S.R Dlouhy, G.H Vance, and P.H. Vogt.(1997). ‘Multiplex PCR: Critical Parameters and Step-by-Step Protocol.’ BioTechniques Vol. 23 pp.504-511. Home: UK Genetic Testing Network. (2001-2008). http://www.ukgtn.nhs.uk/gtn/Home (accessed December 4, 2008). Human Genome Project 92008). Human Genome Project Information. www.ornl.gov/sci/techresources/human_genome/medicine/genetest.shtml (accessed December 3, 2008). Khoury, Muin J, Linda L McCabe, and Edward, R.B. McCabe. (2003). ‘Population Screening in the Age of Genomic Medicine.’ New England Journal of Medicine,Vol. 348 pp.50-58. Kwok, Pui-Yan. (2001). ‘Reflections on a DNA Mutation Scanning Tool.’ Nature Biotechnology, pp. 18-19. Liang, Peng, and Arthur B Pardee.(1992). ‘Differential display of eukaryotic messenger rna by means of the polymerase chain reaction.’ Science, Vol. 257 pp. 967-970. Molecular Genetics Laboratory. (2008) Oxford Radcliffe Hospitals NHS Trust. http://www.oxfordradcliffe.nhs.uk/forpatients/departments/labs/geneticslab/documents/diseaseservices.pdf (accessed December 5, 2008). Watson, J.D, and F.H.C. Crick. (1953). ‘Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid.’ Nature, Vol 171. pp. 737-738. Weissleder, Ralph.(1999). ‘Molecular imaging: exploring the next frontier.’ Radiology, Vol. 212 pp. 609-614. Appendix Table 1. List of diseases, genetic tests and cost of tests at the Molecular Genetics Laboratory of the National Health Service, UK. Read More