GENETICS – GENETIC MUTATIONS – GENETIC INHERITANCE

The majority of human diseases, perhaps with the only exception of infectious diseases, has a genetic basis, or at least a genetic predisposition, even if this is often difficult to detect given that the clinical phenotype (ie, the manifestations of the disease) results from the interaction between individual genetics and the environmental factors (multifactorial diseases). The monogenic diseases are those conditions in which the alteration of a single gene is capable of causing the disease. To understand the basic rules of the transmission of genetic diseases, keeping in mind that our genetic heritage is double is necessary. In other words, we have two copies of every gene, one of which is inherited from the mother and another one is inherited from the father.

In arrhythmogenic genetic diseases the transmission can be:

Autosomal dominant: 50% chance that the disease is transmitted, regardless of the gender.

Autosomal recessive: the disease is clinically present only if the defect is inherited in double dose, ie by both parents. The genetic defect will be, therefore, present in homozygosity. The bearer of a single abnormal gene (heterozygous) can be defined as an healthy carrier of the disease. In a couple where both parents are carriers (heterozygous) you will have 25% chance of having a child who does not have the genetic defect, 25% chance of having a child homozygous (therefore manifesting the disease) and 50% chance to generate a heterozygote (healthy carrier). Autosomal recessive diseases occur more frequently when there is parental consanguinity.

In the reality of the clinical practice, two factors make it more complex to study the inheritance of a disease: the incomplete penetrance and the variable expressivity.

The penetrance is the ratio between the number of individuals who manifest the phenotype and the number of individuals affected; incomplete means that not necessarily all of the carriers of the genetic defect will show the disease. The variable expression is due to the fact that the same genetic defect may manifest differently in different individuals: this is why we have, in the same family, subjects with serious clinical manifestations and people with the disease in a mild form.

HOW WORKS GENETIC ANALYSIS?

DNA EXTRACTION

The first step of a genetic analysis is the extraction of DNA. The DNA is located in all cells of our body, and it can be extracted from any tissue, even if the venous blood represents the most used for clinical purposes, because it represents an easy source of DNA. The only nucleated bloody cells are the white blood cells from which DNA is extracted. The DNA is separated from the protein. The DNA extraction process takes 24-48 hours. The greater the number of genes analysed, the greater will be the quantity of DNA available.
The PCR technique (Polymerase Chain Reaction) is the basic methodology used in genetic and molecular biology to obtain thousands of copies of the DNA fragment that we wish to study. In fact, DNA obtained from blood is usually scarce for the analysis. To get this kind of selective amplification is necessary to “cut” from the total DNA the single fragment of interests, therefore multiply it until reach the right amount needed to perform the analysis. In order to reach this aim, a enzyme (Taq DNA polymerase) together with a device able to rapidly and precisely modify the temperature in the tubes that contain the DNA to be analyzed (the so-called Thermal Cycler) is used.
PCR allows to amplify only short fragments of DNA, but a gene is usually composed of several thousands of base pairs. For this reason many PCR reactions are necessary to analyse a whole gene. Therefore the genetic investigation, especially for diseases in which particular genes have been identified, takes very long and laborious time.

DNA Analysis

The DNA amplified by PCR protocol can be analysed following distinct techniques to look for genetic deficiencies. The most reliable methods are: SSCP (Single Strand Conformational Polymorphism), DHPLC (Denaturating High Performance Liquid Chromatography) and DNA sequencing. The final aim of these techniques is to identify mutations, which are the “errors” in the DNA sequence that cause the disease. DNA (deoxyribonucleic acid) is composed by an extremely long chain of molecules, the basis cytosine, guanine, adenine, and thymine, that carry the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms.

The mutations

As previously described, a gene is made up of a chain of many thousands of DNA sequences. In some cases one single error (for example an adenine in place of a guanine) can cause a genetic disease.
The principle mutations are:
• the mutations in which a single DNA base is ‘incorrect’
• the cases in which there is a missing element of DNA of varying proportions
• the insertions which are due to the abnormal addition of a fragment of DNA in a gene

Furthermore, polymorphisms may be present in the DNA: these are alterations in the genetic sequence present in the general population with a frequence superior to 1%. Some polymorphisms, whilst not causing diseases, could be responsible for a modification of the clinical manifestations.

The pathogenesis

The detection of a mutation in the DNA of a patient with arithmogenic disease helps to better understand the pathogenesis of the symptoms and provide an important element to the doctor in the identification of the most appropriate cure, revealing the stratification of the risks based on the genotype and a gene-specific therapeutic approach. Moreove, the idenfication of the the genetic defect helps the patient’s family, in so far as it reveals the identification of the silent carrier of the disease and therefore makes a prenatal diagnosis possible.

Cardio-genetic Consultation

Cardio-genetic consultation is a direct method of helping the family in which the genetically based cardiovascular disease was identified to face the problems connected to the disease. The consultation is, therefore, an integral part of the genetic test: It should procede based thereon – to give the patient the option of choosing genetic screening, whilst knowing all the personal limitations and possible consequences to them and their family, both good and bad – to guarantee the patient the best possible information on the clinical implications of the diagnosis. Moreover, through the course of the consultation, the patient can freely express their wish to be informed of the results of the diagnosis, or whether to inform their family and take the decision of carrying out an analysis on the potential risk to their children.

Genetic consultation must be carried out by professionals who are specifically trained in the procedures, norms and manners which are different to those used in normal clinical practises. The objective of genetic consultation is to be informative and not directive, providing the patient with the necessary elements they need to make an informed decision.

When and why undergo Genetic Analysis?

If a patient is diagnosed the genetic analysis provides two fundamental pieces of information: the first relative to the patient, the second relative to the patient’s family. In regards to the patient themself, the identification of the genetic variations of the the disease in which various genes may be factors, may help to provide a better stratification of the risks and to address the therapy in a gene-specific way. Once the identification of the genetic defect of the propositus (the first affected individual of a family which adds to the doctors observations), the extension of the genetic analysis to the apparently healthy family or one with an unclear medical case, brings to light the so called ‘silent carriers’, subjects who, even though at the time of diagnosis do not present any relevant clinical problems, which may later develop into a disease. When the diagnosis of a disease is only suspected, the identification of a mutant gene renders it certain.

In depth studies at a Clinical Level

It is fundamentally important to remember that in many monogenic diseases only a few of the diseased genes have been identified: This limit ensures that the missing identification of a mutation does not allow for the exclusion of the diagnosis of a disease.
For this reason the patient who undergoes genetic analysis must be previously thoroughly studied at a clinical level. It is for this that the importance, for the clinic who carries out the genetic analysis, to have access to all the information in correlation to the clinical information relative to the patient: necessary in order to avoid inappropriate investigations or analysis of genes that are not relevant to the clinical picture of the patient.
It is possible to carry out a prenatal diagnosis of the members of a family in which the genetic defect responsible for the disease has been identified. Prenatal diagnosis, in its delicate role, must always proceed with an accurate genetic consultation, with the aim of being informative as opposed the results of the analysis being a benefit to others.

Research

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Statics

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