Medical Genetics - V. M. Zaporozhan 2005

Methods of Diagnostics of Hereditary Diseases
Molecular Genetic Methods (DNA Diagnostics Methods)
Direct and Indirect Methods of DNA Diagnostics

Molecular-Genetic Methods are divided into direct and indirect methods.

Direct Diagnosis involves the detection of a mutation in the Gene under study. It has virtually absolute accuracy, requires only a DNA sample from the examined individual for analysis, and can be performed in both familial and sporadic cases of diseases. Conducting direct DNA diagnosis requires precise knowledge of the gene Structure (or the specific gene region containing the analyzed mutation). Searching for disease-causing Mutations is a challenging task, as a condition can be caused by various mutations in the same gene. However, as a rule, a specific type of mutation is more prevalent in a given population. Mutations that predominate and account for a significant percentage of all disease cases in a given population are called major mutations. For instance, most cases of phenylketonuria are caused by the R408W mutation in the disease-associated gene; cystic fibrosis is most commonly caused by the AF508 mutation in the chloride channel protein gene; trinucleotide repeat expansions are responsible for fragile X syndrome, deletions for Duchenne muscular dystrophy, and extended deletions for congenital adrenal hyperplasia, etc. Knowledge of major mutations facilitates direct DNA diagnosis.

The methodological approaches used in direct DNA diagnosis of a particular hereditary disease depend on The Nature of the mutations and the Molecular Organization of the corresponding gene.

PCR-based methods are widely used in direct Diagnostics. By applying primers to the gene region where a mutation most frequently occurs, A large number of copies of this region are obtained. Further Analysis of the PCR products depends on the type of mutation. For example, if the disease is caused by nucleotide deletions, duplications, or trinucleotide repeat expansions, the amplified fragments of the normal and mutant genes will differ in length and electrophoretic mobility. In this case, following Electrophoresis of the fragments in an agarose gel, restriction fragment length polymorphism (RFLP) analysis is performed (see Fig. 10.8). To detect nucleotide substitutions in the analyzed gene region, sequencing of the amplified fragments becomes necessary. Other techniques can also be used to detect mutations. In some cases, mutations disrupt restriction sites for specific restriction Enzymes. A mutant gene may lack a restriction site for a particular enzyme or, conversely, a restriction site may appear at an unusual Location. In both scenarios, the mutant and normal PCR products will yield restriction fragments of different lengths, which can be easily detected via electrophoresis (RFLP — restriction fragment length polymorphism).

Direct diagnosis utilizes various PCR modifications as well as Other DNA Diagnostic techniques, such as Southern blot Hybridization, in vitro protein product Translation, etc.

Indirect diagnosis is used for conditions where the gene has been mapped (its chromosomal localization is known), but the Gene Structure and the nature of its mutations are not yet sufficiently understood. The Essence of indirect DNA diagnosis lies in analyzing the inheritance of polymorphic genetic markers—linked to the disease gene (i.e., located at neighboring loci on the chromosome and inherited together according to Morgan's laws)—in affected and unaffected family members. Such markers may include restriction sites for specific restriction enzymes, as well as polymorphisms of minisatellite and microsatellite sequences.

Restriction sites can be located adjacent to the pathological gene in non-coding DNA regions. Human DNA is characterized by single-nucleotide polymorphism—non-coding DNA regions frequently contain single-nucleotide substitutions and deletions. These may affect restriction sites linked to the pathological gene, thereby altering the length of The DNA Restriction fragments (restriction fragment length polymorphism).

Microsatellite DNAs are short tandem repeats consisting of 2–6 (most commonly 2–4) NUCLEOTIDES. The most prevalent are dinucleotide CA-repeats. They are arranged in tandem clusters (i.e., groups following one another, e.g., CACACA....). Tri-, tetra-, and pentanucleotide tandem repeats, known as STRs (short tandem repeats), are also common. The number of repeats at the same locus can vary significantly among different individuals; consequently, the cluster lengths are polymorphic. Because microsatellites are predominantly located in non-coding DNA regions, Changes in the number of repeats are not phenotypically expressed. They are inherited according to Mendelian laws: a child receives one chromosome from the mother with a specific number of repeats, and the second chromosome from the father with a different number of repeats. If such a microsatellite cluster is located adjacent to or within a gene responsible for a monogenic disease, a specific number of repeats and cluster length can serve as a marker for the pathological gene. In this case, diagnosis is based on analyzing the length of short tandem DNA repeats (STRs).

Minisatellite DNAs are tandem repeats of a larger number of nucleotides (15–100). They are known as VNTRs (variable number tandem repeats). The length of these loci also varies significantly among individuals and can serve as a marker for a pathological gene. DNA frequently contains clusters of minisatellite tandem repeats of 10–15 nucleotides, which were first discovered by the English geneticist Jeffreys. They are used less frequently than microsatellite DNAs.

A limitation of indirect methods is the mandatory preliminary Study of the genotype of at least one affected relative in order to establish the linkage of the pathological gene with a specific polymorphic marker. In the absence of affected relatives available for examination, diagnosis is generally impossible. Furthermore, indirect DNA diagnosis always carries a risk of error associated with potential meiotic recombination between the disease gene and the studied marker, As a result of which the “pathological” marker allele and the disease gene may segregate onto different Chromosomes in the offspring. The magnitude of such an error is directly proportional to the distance between the marker locus and the gene under study: the closer they are on the chromosome, the lower the probability of Separation due to Crossing-over. For currently known genetic markers used in indirect DNA diagnosis of Hereditary diseases, the probability of crossing-over with the disease gene does not exceed 1–5%. Thus, the accuracy of indirect DNA diagnosis is typically 95% or higher.

Indirect methods can also be applied to diseases whose genes have been identified but are characterized by a large size and complex molecular organization, making direct mutation detection difficult. Examples of such disorders with deciphered genes for which indirect DNA diagnosis is used include ataxia-telangiectasia, Wilson's disease, etc.