Human Biochemistry Volume 2 - Murray R. 1993

Structure, Function, and Replication of Information Macromolecules
Recombinant DNA Technology
Genetic Engineering and Analysis of the Molecular Basis of Diseases

Normal Genetic Variants

Normal variants of human DNA sequences (polymorphisms) do exist. Such variants occur roughly once every 500 NUCLEOTIDES, or 10⁷ times per genome. They include deletions, insertions, and single nucleotide substitutions. In healthy individuals, these alterations either avoid coding sequences altogether or occur within functionally insignificant regions of DNA coding areas. Introduction/20.html">DNA Structure polymorphism can also be directly linked to specific diseases. In recent years, The phenomenon of polymorphism has been increasingly utilized to identify their corresponding specific genes.

Disease-Causing Genetic Alterations

At earlier stages in The Development of medical genetics, the prevailing view was that most Hereditary diseases were caused by point Mutations manifesting as the functional imperfection of a corresponding altered protein. The Role of point mutations in the onset of hereditary pathologies is indeed significant; however, it should be added that Genetic Disorders can be triggered by disruptions at any stage of the process illustrated in Fig. 36.1. This principle is well illustrated by studies of the ß-globin Gene, which features a clustered Organization and has been mapped to the eleventh chromosome (Figs. 36.7, 36.8). The Biosynthesis of defective ß-globin causes a wide range of diseases. Pathological symptoms can stem from abnormalities either within the gene itself or in its flanking environment (Table 36.6).

Point Mutations

A classic example of a disease associated with a point mutation is Sickle-Cell Anemia. The condition is triggered by the substitution of just a single base out of the 3×10⁹ bases that comprise the complete Human Genome: in the sixth codon of the ß-globin gene, adenine is replaced by thymine. Instead of glutamic acid, valine is encoded, leading to structural abnormalities in the ß-globin molecule. Some point mutations cause a reduction or complete cessation of ß-globin synthesis. The result of such disruptions is ß-thalassemia (thalassemias being a Class of disorders associated with impaired globin synthesis). Figure 36.8 illustrates the positions of point mutations that disrupt any of the numerous stages involved in producing normal ß-globin mRNA, thereby causing ß-thalassemia.

Fig. 36.8. Mutations of the ß-globin gene responsible for ß-thalassemia. The gene is shown in the 5' → 3' orientation. The untranslated 5'- and 3'-regions are hatched. When read in the 5' → 3' direction, the dark regions represent exons 1–3, and the light regions represent introns 1 and 2. Mutations affecting METABOLISM/31.html">Transcription control (●) are localized in the 5'-flanking region. Several nonsense mutations (∆), mutations affecting RNA Processing (◊), and splicing mutations (O) indicated in the figure have been identified. A large number of mutations have been detected in certain regions; such areas are marked with square brackets.

Table 36.6. Structural Alterations in the ß-globin Gene

Deletions, Insertions, and DNA Rearrangements

Studies of bacterial, viral, Yeast, and Drosophila genomes demonstrate that specific DNA segments can change their position within The Genome. The loss of a functionally important DNA region, the rearrangement of DNA fragments within a gene, or an insertion into its coding or regulatory region typically alters the expression level of that gene, leading to disease. Molecular analysis of ß-thalassemia reveals a large number of such cases (particularly deletions). It appears that globin gene clusters are highly susceptible to damage. Deletions within the a-globin cluster, localized on the 16th chromosome, lead to a-thalassemia. For most of these deletions, a clear correlation with ethnic origin has been established. Northern Europeans, Filipinos, Negroids, and Mediterranean populations exhibit various types of abnormalities, yet all of them lead to the loss of hemoglobin A and a-thalassemia.

A similar analysis can be performed for many other diseases. Point mutations are typically identified by determining The nucleotide sequence of the gene under study, but if a mutation affects a restriction site, restriction analysis is sufficient for its detection. Deletions and insertions of DNA fragments larger than 50 Base Pairs are identified using Southern blotting.

Pedigree Analysis

The example of sickle-cell anemia clearly demonstrates how effective the Genetic Engineering approach is for studying human diseases. A base substitution in the coding strand of the hemoglobin gene alters the sequence corresponding to the sixth codon:

This abolishes the Mst II restriction site (CCTNAGG; Cleavage sites are indicated by arrows, see Table 36.1). Other Mst II sites (Fig. 36.9) remain intact and can be cleaved by this restriction enzyme. Therefore, Southern blot analysis of enzyme-treated DNA samples from normal (AA), heterozygous (AS), and homozygous (SS) patients yields three distinct fragment distribution patterns (Fig. 36.9). This example shows how pedigree analysis can be conducted at the DNA level using the principles and approaches described in this chapter. Such an analysis is highly effective in studying a range of genetic disorders caused by deletions, insertions, and (more rarely) point mutations that alter restriction sites (as in the case just considered).

Fig. 36.9. Pedigree analysis in sickle-cell anemia. The top part of the figure (A) shows the beginning of the ß-globin gene with Mst II restriction sites (T) in normal (A) and sickle-cell (S) ß-globin. Digestion of healthy individuals' DNA with the restriction enzyme Mst II generates specific DNA fragments of 1.15 and 0.2 kb in size. A single base substitution in sickle-cell anemia patients results in the loss of one of the three Mst II sites in the gene region, and accordingly, the appearance of a single specific Mst II fragment 1.35 kb in size. This difference in length is easily detected by Southern blotting (B). (THE POSITION OF the 0.2 kb fragment is not indicated in this figure.) Pedigree analysis demonstrates three possible genotypes: AA normal (O), AS sickle-cell trait heterozygote (◑◨), and SS sickle-cell homozygote (■). This approach enables Prenatal Diagnosis of sickle-cell anemia and the identification of heterozygous carriers of the corresponding gene (◆).

Prenatal Diagnosis

Prenatal diagnosis of hereditary disorders is feasible if The Nature of the genetic defect is known and an appropriate probe is available. Southern blot analysis can be performed on DNA extracted from Cells collected from 10 ml of Amniotic Fluid (or obtained via chorionic villus sampling). A fetus with the AA restriction variant (Fig. 36.9) is normal and does not carry the sickle-cell trait. In the case of the SS variant, the Development of the disease can be predicted with certainty. Probes for performing such analyses are already available for many diseases.

Restriction Fragment Length Polymorphism (RFLP)

DNA sequence alterations caused by the factors described above can alter the arrangement of restriction sites and consequently affect restriction fragment lengths. A stable, heritable change in the distribution of restriction fragment lengths (observed in more than 1% of the population) is referred to as restriction fragment length polymorphism (RFLP). This phenomenon can result either from point substitutions (sickle-cell anemia) or from deletions and insertions (thalassemias). Recently, RFLPs have been successfully employed for diagnostic purposes. Restriction polymorphism has been discovered both within known gene sequences and in DNA regions of unknown function. An RFLP may disrupt biological function or have no biological consequences whatsoever; in either case, the corresponding altered loci are inherited in a Mendelian fashion. The primary application of this phenomenon (with approximately 350 RFLPs already known) is the diagnosis of hereditary diseases whose functional nature is unknown. First, an RFLP is used to establish a linkage group, and then the disease-responsible locus is determined via sequential Hybridization. According to the sequential hybridization method, a fragment representing one end of a long DNA strand is used as a probe to identify an overlapping yet extended adjacent fragment. Applying this approach, known as chromosome walking (Fig. 36.10), enables "stepping" along the DNA strand until the region of interest is reached. The direction of movement is monitored using a restriction map. Chromosome walking is particularly convenient for studying X-linked diseases, since only one of the two alleles is expressed in this case. 20% of identified RFLPs belong to the X chromosome, for which a virtually complete map has been constructed. Utilizing the RFLP phenomenon allows for the localization of the gene for any X-linked disease (e.g., Duchenne muscular dystrophy). RFLP analysis has helped establish that the genetic defect in Huntington's chorea affects the end of the short arm of chromosome 4, while the gene causing Polycystic Kidney Disease is linked to the a-globin locus on chromosome 16.

Gene Therapy

Diseases caused by the functional deficiency of a particular gene product can be treated using "replacement" therapy (Table 36.5). The strategic approach involves cloning the gene (e.g., the gene encoding adenosine deaminase) into a vector capable of integrating into the host cell genome. Using Bone Marrow cell precursors for this purpose holds great promise. It is hoped that such cells will "engraft" and proliferate, synthesizing the transgenic product. Of course, a gene transferred into somatic cells is not transmitted to offspring.

In recent years, intensive efforts have been underway to explore ways of applying genetic engineering to Germ Cells. Relevant experiments are being conducted on laboratory animals. Genes injected into fertilized mouse oocytes are, in some cases, integrated into the host genome. The resulting Transgenic Animals are utilized to study patterns of Gene Expression across various Tissues and to identify specific genes involved in ontogenesis. Recently, the transgenic approach was successfully applied to correct a genetic defect in mice. DNA containing the coding sequence for the gonadotropin-releasing hormone precursor was injected into fertilized oocytes of mice with hereditary hypogonadism (Ch. 55). In some of the mice that developed from these oocytes, the gene was expressed normally. Phenotypically, these mice were normal in every respect. Their offspring also showed no deficiency in gonadotropin-releasing hormone. This example demonstrates the feasibility of transgene expression in somatic cells and its transmission to subsequent generations.

Fig. 36.10. Chromosome walking method. Suppose it is necessary to locate gene X within an extended DNA fragment. The exact position of the gene is unknown, but a primary probe (*) is available that corresponds to a certain region of the genome (shown here at the 5' end of the DNA fragment under study). In addition, a library of overlapping genomic fragments is available. (For simplicity, only five fragments are shown in the figure.) The primary probe hybridizes only with clones containing fragment 1. This fragment can subsequently be used as a probe to identify fragment 2. The sequential hybridization Procedure is repeated until fragment 4 is found, which hybridizes with fragment 5 containing the target gene X.