The exome is the part of the genome (set of DNA molecules) formed by exons, the fragments of DNA that are transcribed to give rise to proteins, the non-coding parts are called introns. The study of exome is one of the most complete and complex ways to study our DNA.
A gene is the unit of genetic material that provides the information necessary for the synthesis of a protein. A gene is formed by a long chain of nucleotides, in which exons and introns are distinguished.
The exons are the coding regions that will provide the information for the synthesis of a protein, while the introns are non-coding regions, which are interspersed in the gene and have other functions.
An intron is a region of DNA that is part of the primary transcript of RNA, but unlike exons, they are removed from transcription, prior to translation, they would be like the semicolons an de dots of a text.
The introns are eliminated from the sequence of messenger RNA by the process called alternative splicing, which encompasses both the removal of the intron by specialized proteins and the subsequent binding of exons that are loose.
Codons that code for the same amino acid often have the first two nucleotides the same, changing only the third. Thus, changes in the nucleotide of the third position do not involve changes in the amino acid (silent mutations).
An anticodon is the sequence of three nucleotides complementary to a sequence of three other nucleotides found in messenger RNA (mRNA), the latter being the codon. The anticodon, on the other hand, is part of one end of a transfer RNA molecule (tRNA).
In this way, the impact of point mutations when they occur in the third position of the codon is minimized. In contrast, mutations in the first and second position of the codon usually involve an amino acid change (missense mutations).
The human exome consists of approximately 180,000 exons that make up about 1% of the total genome (about 30 megabases of DNA).
The study of the exome may be necessary to arrive at a genetic diagnosis, which is a medical tool that allows to determine the genetic causes of a disease.
-Importance of Exoma:
Our understanding of the genetics of colorectal cancer has changed dramatically in recent years. Colorectal cancer can be classified in different ways. Along with the advent of complete exome sequencing, we have gained an understanding of the scale of the genetic changes found in sporadic colorectal cancer.
Now we know that there are multiple pathways that are commonly involved in the evolution of colorectal cancer, such as, RAS, and kinase, among others. Another recent advance in our understanding of the genetics of colorectal cancer is the recognition that many, if not all, tumors are actually genetically heterogeneous within individual tumors.
Recent research has revealed the prognostic and possibly therapeutic implications of several specific mutations, including specific mutations in BRAF and KRAS. There is growing interest in the use of mutation tests for screening and surveillance through stool and circulating DNA tests.
Advances in translational research in colorectal cancer genetics are radically changing our understanding of colorectal cancer and will likely change therapy and surveillance in the near future.
The diseases have clinical characteristics that determine them as such, differentiating them from another disease. Many diseases have a genetic basis, that is, the disease is due to mutations in a certain gene. A genetic diagnosis consists in detecting the gene and the mutations in it that cause the disease.
-What are mutations?
A mutation is a change of base pairs in the DNA sequence with respect to the original sequence, a codon is a triplet of nucleotides. In the genetic code, each amino acid is encoded by one or more codons and each codon carries the information to pass to the nucleotide sequence of the mRNA (messenger RNA), to the amino acid sequence of the protein in the translation process.
It must be distiguished of a chromosomal translocation, which means that the chromosome has been broken, which allows its union with other chromosomal parts. In Burkitt’s lymphoma, it affects chromosome 8 (locus of the Myc gene), which changes the expression pattern of the Myc gene, altering its natural control function in cell growth and proliferation.
So we must also consider the numerical mutations: Individuals with a numerical chromosome variation have one or more chromosomes of more or less than the normal chromosomal complement, as is the case of Down’s Disease.
We all inherit 23 chromosomes from each of our parents that contain long DNA molecules. Mutations occur in all of us. These are small changes, where one or two base pairs do not align correctly during the early phase of cell division, which helps make each of us unique.
Most of the genetic changes are located in regions of the DNA that do not imply a biological impact, that is, they do not alter the normal function of our organism. But sometimes the mutation occurs in a functional gene and causes problems.
These problems are those that cause diseases of genetic base. The new generation sequencing allows to know all the coding genes.
Two methods, the complete sequencing of the exome and the complete sequencing of the genome, are increasingly used in clinical and research to identify genetic variations, both methods are based on new technologies that allow the rapid sequencing of large amounts of DNA. These approaches are known as next-generation sequencing (or next-generation sequencing).
With the name in English Next Generation Sequencing, usually refers to new techniques of massive sequencing that allow us to know the information found in our genome (all our DNA), quickly and increasingly expeditiously.
The original sequencing technology, called the Sanger sequence (named after the scientist who developed it, Frederick Sanger), was a breakthrough that helped scientists determine the human genetic code, but it is slow and expensive.
Sanger’s method has been automated to make it faster and is still used in current laboratories to sequence short pieces of DNA, but it would take years to sequence all of a person’s DNA (known as the person’s genome)
Next-generation sequencing has accelerated the process (taking only days or weeks to sequence a human genome) and reduces the economic cost. This method allows to identify variations in the coding region of proteins of any gene, instead of only in a few selected genes.
Because most of the known mutations that cause the disease occur in exons, it is believed that complete sequencing of the exome is an efficient method to identify possible mutations that cause disease.
It is a sequencing technique (to know the order in which the DNA bases are grouped, that is, how all the coding genes are ordered in the DNA chain. Also known as WES (whole exome sequencing)
It consists in selecting first that subset of DNA that encodes the proteins, that is, the exons. In a later step, the aim is to identify the sequence using a high-throughput DNA sequencing technology or massive sequencing.
Mass sequencing techniques have experienced great advances in recent years. The first one, and fundamental for the interpretation of the results of these studies, was the completion of the Human Genome Project in 2003.
It was an international project that began in 1990 with the aim of determining the sequence of pairs of chemical bases that make up human DNA, and of identifying and mapping all the genes of the human genome, both from a physical and functional point of view. It has been the largest international collaborative biological project in the world.
At that time it is estimated that the complete sequencing of the genome had a cost of more than 2 billion dollars. However, thanks to the advancement of technology, both the time for the study of the exome and the associated cost has been decreasing, being about 200,000 US dollars in 2008 and 10,000 dollars in 2010.
Now, we only talk about the cost of the technological part, since there is an essential and very important part of interpretation of the amount of data that these techniques provide, which represents an extra cost.
Usually our exome contains hundreds of random changes that do not always have a clinical repercussion (they do not always cause illness). Therefore it is essential to filter those changes that we find to know which of all the findings would explain the disease we are trying to diagnose.
For this, we resort to the study of parents’ exome or to the analysis of the functions of the genes where the changes are found, among others. This is giving rise to the need to use the knowledge of an emerging discipline, bioinformatics, to help the clinician or the geneticist to interpret the results.
When an exome study is carried out, both the patient (index case) and the parents are studied to help interpret the findings. When a change that can be pathological is detected, Sanger sequencing is performed to confirm it (the same as performed routinely for the study of a specific gene).
Many times, it is also necessary to study healthy family members (brothers or sisters of the index case) to confirm or rule out a change as a mutation.
Knowing which gene is mutated in each patient gives a genetic confirmation to the clinical diagnosis made by the doctor. This allows the genetic counseling of a hereditary disease.
It is thought that in the not too distant future it will be like this, and the drugs used to treat certain genetic diseases will be related to the mutated gene, the type of mutation, as well as the clinical presentation of the patient.
It has been discovered that DNA variations outside the exons can also affect gene activity and protein production and lead to genetic disorders, variations that the complete sequencing of the exome would not detect, this would be the case of epigenetics.
Due to the ternary nature of the genetic code understood as a succession of codons, the insertion or deletion of a number of nucleotides not divisible by three, can change the reading frame of the gene, causing a completely different translation to the original. The sooner the insertion or deletion appears in the gene, the greater the alteration that the protein undergoes.
A reading frame mutation is not the same as a simple nucleotide polymorphism, in which the replacement of a single nucleotide occurs, rather than being lost or gained.
A mutation-shift reading frame can, in general, lead to the reading of codons in the sequence subsequent to the mutation and code for different amino acids.
The reading frame mutations appear in several genetic diseases such as cystic fibrosis, increase the susceptibility to certain types of cancer and some types of familial hypercholesterolemia. In 1997, the relationship between a reading frame mutation and resistance to HIV infection was established.
The frame shift may also cause the appearance or disappearance of a termination codon (UAA, UGA, or UAG) in a position different from the sequence. The created polypeptide is then abnormally short or too long, and in most cases loses its functionality.
In clinical practice its use is limited, although each time you are having more accessibility to these techniques. It is usually resorted to faster techniques, cheaper and easier to interpret, such as the study of a single gene (guided by biochemical suspicion and clinic) or the use of gene panels for a group of genes.
The criterion for choosing the most appropriate genetic study method should be determined by the close collaboration between the clinicians, biochemists and geneticists who care for the patient.