Basics of Medical Genetics - Buzhiyevska T.I. 2001

Medical aspects of genetic engineering biotechnology

Recent breakthroughs in general, medical, and particularly Molecular Genetics have provided a fresh perspective on the Etiology AND Pathogenesis of diseases, tracing them back to the trait-coding molecule itself. The term molecular medicine is being used with increasing frequency. Modern scientific insights and the capabilities of Molecular Diagnostics—at the level of either the Gene or its product—necessitate the Application of Molecular therapy. Worldwide, specialized companies industrially manufacture and market diagnostic molecules, such as specific Enzymes (DNA Restriction Endonucleases, ligases, reverse transcriptases, etc.) that target specific nucleic acid sequences, synthesize DNA from DNA or RNA templates, and cleave DNA molecules at precisely defined sites_; generating fragments of the desired length. This forms The basis of restriction fragment length polymorphism (RFLP) analysis, a molecular diagnostic method used to detect the carrier state of Mutations linked to specific DNA fragments. Developed in the late 1970s and early 1980s, this method is now widely used for both gene mapping and the molecular Diagnosis of hereditary disorders.

By the late 1980s and early 1990s, over 300,000 human genes had been mapped. Scientists in the United States have led the Human Genome Project with the greatest success (mapping 50% of genes), followed by the United Kingdom (15%). Russian scientists have also contributed significantly to this initiative: S. A. Limborska decoded several sequences on the X chromosome; V. A. Evgrafov mapped the gene responsible for Friedreich's ataxia; E. K. Ginter mapped the keratin gene associated with palmoplantar keratoderma; and D. V. Zaletaev mapped the gene for tricho-rhino-phalangeal syndrome type II (Langer-Giedion syndrome).

In Ukraine, such research is spearheaded by scientists at the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, most notably V. M. Kavsan (mapping expressed genes).

The methodologies for gene mapping and DNA-based mutation diagnostics are fundamentally similar, consisting of the same core stages: cloning target DNA sequences, isolating DNA from a patient's Cells, DNA Restriction Digestion, and Hybridization.

The ability to synthesize in vitro polynucleotides complementary to a specific DNA region underpins another powerful technique: the Polymerase Chain Reaction (PCR). PCR enables the detection of specific mutations within a haplotype, the diagnosis of homozygous or heterozygous carriage of a pathology at any stage of ontogenesis, and the analysis of any cells in the body.

PCR is a DNA technology of immense practical value. It allows for the selective Amplification—by a factor of a billion—of a specific DNA segment ranging from 1 to 20,000 Base Pairs in length. To target a specific segment, 15–20 nucleotide DNA primers are required that are complementary to the sequences flanking the region of interest. These primers and a thermostable DNA polymerase are added to the patient's DNA sample. Upon heating, the patient's DNA strands separate; during the subsequent cooling phase, the primers bind complementarily to the ends of the target fragment, and the DNA polymerase synthesizes a complementary strand between them. The mixture is then reheated to separate the newly formed double helix, cooled again to allow the two strands to act as templates for another round of synthesis, and so on. As a result, a DNA segment that would be entirely undetectable within the whole genome is amplified over thirty thermal cycles into a quantity of DNA sufficient for detailed study and even sequencing (i.e., reading The nucleotide sequence).

Using PCR, clinicians and researchers can genotype samples to: 1) detect integrated viral sequences (papillomavirus, HUMAN IMMUNODEFICIENCY VIRUS, hepatitis virus, cytomegalovirus); 2) identify oncogenes; 3) detect mutations causing hereditary disorders in the homozygous state (cystic fibrosis, phenylketonuria, Sickle Cell anemia, etc.), heterozygous state (Huntington's disease, neurofibromatosis, etc.), hemizygous state (X-linked Duchenne and Becker muscular dystrophies, hemophilia, etc.), or predisposing to atherosclerosis and coronary artery disease. This method enables prenatal diagnosis prior to the clinical onset of symptoms, thereby expanding the possibilities for preventive care, the creation of an adaptive environment, and personalized therapy for the individual patient.

Diagnostic molecules—such as primers, probes, and various binding assays—are produced via Introduction/32.html">Genetic Engineering BIOTECHNOLOGY as commercial diagnostic kits complete with price lists and order forms; their widespread clinical use is limited only by their high cost.

Diagnosing diseases at THE MOLECULAR LEVEL completely transforms the therapeutic approach. Understanding that a condition is caused by a mutation in a specific gene guides Treatment toward administering functional products of the normal gene (indirect Gene Therapy) or even introducing the normal gene itself into the patient's cells (direct gene therapy, sometimes referred to as Gene Surgery).

While molecular diagnostics and the bioengineering of diagnostic molecules raise no ethical concerns, gene therapy—built on the principles of intervening in Genetic information and creating novel organisms—brings forth serious new deontological dilemmas.

Among therapeutic molecules, Hormones—signaling molecules with broad regulatory Functions—are of paramount importance. They govern numerous bodily processes and regulate the harmonious execution of the genetic program. Deficiencies or defects in hormones caused by specific mutations lead to severe endocrine disorders which, like all Hereditary diseases, require lifelong molecular therapy. Insulin, vital for treating Diabetes Mellitus—the most common endocrine disorder, affecting up to 25.5% of the population in various groups—has been sourced from the pancreases of cattle and pigs since 1922. Producing 100 grams of crystalline insulin requires the pancreases of 4,000 cows. Chemical synthesis of insulin involves 170 distinct Chemical Reactions, making industrial production extremely difficult. In 1979, insulin synthesis via genetic engineering began, proving remarkably productive: in the UK, gene-engineered synthesis yields 100 grams of insulin from 1,000 liters of culture broth, replacing the 800 kg of pancreatic tissue previously harvested from 4,000 cows.

Genetically determined Growth Hormone deficiency causes Pituitary dwarfism, which occurs in 1 in 5,000 children. Because this hormone is species-specific, patients require human somatotropin. Prior to the era of recombinant synthesis, this was obtained from the pituitary glands of human cadavers, supplying enough material to treat only one-third of those in need. Consequently, there is no doubt about the immense advantages of using genetic engineering to produce human somatotropin in Bacteria into which the human growth hormone gene has been introduced. According to the Swedish firm Kabi Vitrum, a manufacturer of the product, just 7 hours of bacterial culture in 1 liter of specific medium yields an amount of somatotropin equivalent to that extracted from the pituitary glands of 60 human cadavers.

Genetic engineering also enables The production of transgenic bacteria carrying the genes for thymic hormones, Brain beta-endorphins, somatotropin-releasing factor (SRF), and thyrotropin-releasing hormone (TRH) from the Hypothalamus.

Recombinant biotechnology is also used with great success to produce interferons, interleukins, and other immunopoietic modulators.

Because the genetic programs of living organisms within our biosphere share a common language and universal laws, the potential for creating hybrid genomes and genotypes is practically limitless. Human genes can be transferred into the genomes not only of bacteria but also of eukaryotes, including Yeast, plants, insects, and animals. Scientists are actively working on creating Transgenic Plants that produce human protein products, such as wheat carrying interferon genes. Agricultural animals (such as cows and sheep) can similarly serve as products of genetic engineering, secreting human hormones, immunomodulators, or other therapeutic Proteins in their milk for the replacement therapy of hereditary defects.

The efficiency of successful gene transfer between different genomes is far from 100%. In various experiments across different model systems, it ranges from 2 to 10%. This necessitates the propagation of transgenic organisms to obtain them in sufficient quantities. This applies primarily to sexually reproducing organisms, as reproduction triggers Mendelian laws—namely, segregation in the progeny. These laws can be bypassed by cloning offspring from somatic cells of a transgenic Organism. Beginning in 1995, Ian Wilmut and his colleagues in Edinburgh, Scotland, worked on cloning a sheep (after 277 attempts) and successfully produced a single ewe, Dolly, using an enucleated oocyte and a mammary gland Cell Nucleus from a single donor sheep. Although she gave birth to offspring, she cannot truly be called a "mother" in the traditional sense (due to the absence of a biological father). Because all chromosomal and cytoplasmic DNA in this cloned offspring originates from a single source, the clone must be an exact genetic copy of the donor. The enucleated oocyte, injected with a diploid somatic nucleus, received the necessary energetic stimulus to begin division and, following the genetic program, developed into Dolly the sheep. In 1997 in the UK, another sheep named Polly was produced via cloning; Polly differs from Dolly in that she is a copy of a transgenic sheep, meaning the donor's genotype—and consequently Polly's—contains the gene for a specific human protein.

The milk of such animals acts as a pharmaceutical (indirect gene therapy) for specific Genetic Disorders. Current efforts are focused on generating large numbers of identical embryos (effectively "monozygotic" twins, if an oocyte injected with a somatic nucleus can be termed a "zygote"). Australian scientists are making fruitful progress in this direction. They divide blastocysts at a specific developmental stage into 30 separate embryos, subsequently growing cells from each blastocyst part and dividing them again prior to the stage of embryonic Cell Differentiation into 30 distinct portions, allowing propagation to proceed in a geometric progression. To date, no researcher has obtained more than 100 embryos from a single blastocyst, yet Australian scientists have already produced approximately 500 bovine embryos (Jan Anderson, 1997). The next step is to successfully raise healthy cattle from these embryos. Such technology promises to be significantly more efficient than Artificial Insemination for livestock propagation.

As always, new scientific breakthroughs are being co-opted (negatively) by minority interests seeking to apply direct Gene cloning and therapy to humans. Eugenic dreams and associated agendas are resurfacing, yet all anti-eugenic arguments and considerations remain identical to those presented in the "Medical and Genetic Counseling" chapter of this manual.

Indirect gene therapy—that is, treatment utilizing a recombinant protein product—cannot be considered entirely ideal. Hereditary defects represent serious, lifelong pathologies, and consequently, correction should ideally be lifelong as well.

Integrating intact genes into the genotype of a patient's cells would make it possible to correct the genetic defect while bypassing the transplant Immunity that complicates Organ and tissue transplantation. The principle behind Human Gene Therapy for hereditary disorders mirrors that of engineering microbial strains to produce biologically vital substances. In these cases, the gene encoding the necessary protein is introduced into the patient's cells. Achieving this requires the gene itself, a delivery vehicle (vector), and recipient cells. All three requirements have been fulfilled both theoretically and practically. The gene can be isolated from the DNA of other organisms, synthesized chemically, or—more simply—synthesized from a Messenger RNA template isolated from competent, differentiated cells of a healthy organism. The DNA must be protected from enzymatic degradation and successfully transported to the site of genomic integration within the target cells. This is accomplished by constructing recombinant circular molecules in which the therapeutic gene is combined with fragments of certain viral genomes. Oncoviruses are often preferred because they readily enter cells and their nuclei, integrating smoothly with the recipient cell's DNA. Various Methods exist for introducing recombinant molecules: DNA carriers (alongside Viruses, salmon sperm DNA is frequently used) combined with calcium-phosphate coprecipitation, encapsulation within erythrocyte ghosts, or incorporation into Liposomes. Alternatively, DNA can be microinjected directly into cell nuclei under visual guidance using automated microinjection systems—a preferred approach for genetic engineering manipulations involving Germ Cells, zygotes, and early embryos.

This exact methodology is used to produce Transgenic Animals carrying integrated foreign genes in all somatic cells, such as mice harboring the rat or human somatotropin gene, or the sheep carrying a human protein gene from which Polly was cloned in the UK in 1997. Concurrently, scientists worldwide are concerned about the potential adverse risks of intervening in The Human Genome from both methodological and ethical standpoints: disruption of genetic information, induction of unwanted mutations, unexpected regulatory alterations, and the reactivation of dormant, potentially dangerous viruses. These questions remain unanswered, leaving only hypotheses and speculation. Could the bovine spongiform encephalopathy ("mad cow") epidemic that broke out in the UK in 1997 be a consequence of genetic engineering experiments?

Japanese scientists conducted experiments introducing the gene for a rabbit membrane protein into mouse eggs. Out of 28 Transgenic Mice, only 3 expressed the gene. Others exhibited developmental abnormalities, while one homozygous transgenic mouse developed premature Aging syndrome alongside age-associated pathologies: Infertility, atherosclerosis, Skin atrophy, Osteoporosis, pulmonary emphysema, and a shortened lifespan. The authors named this novel gene with its distinct phenotypic manifestation *klotho*. This mutant allele is inherited in a Mendelian autosomal recessive manner (Makoto Kuro-O, 1997).

Our research demonstrated the mutagenicity of recombinant molecules in mammalian cells at both the chromosomal and gene levels, with a frequency an order of magnitude higher than that of standard gene transformation.

Given the biological, medical, and ethical challenges associated with direct human gene therapy, the majority of physicians, sociologists, religious figures, legal experts, and medical geneticists advocate for amending the European Convention on Human Rights to explicitly include the right to genetic integrity and the safeguards necessary to enforce it. Most developed nations have already enacted legislation prohibiting human cloning.