Understanding the Basics of Genetics for Molecular Therapeutics

Ever since the decoding of full genome, researchers could link new and existing diseases to specific genes. It took 13 years to generate the first sequence of the human genome. Now, genome sequencing of an individual can be done within a day. Advanced healthcare in the developed nations is looking more into identifying specific genes, proteins, and other cellular elements of disease causation. It requires collaborative effort of inter-disciplinary expertise of health science, genetic engineering, data analysis, biostatistics, and bioinformatics to filter the enormous data that is now being generated from gene sequencing. Before delving into the of the world of genomics, a recap of the basic science of genes might help readers register the magnificent therapies that are already out there to provide tailor made patient management.

What is the molecule of life?

Deoxyribonucleic acid (DNA) is the molecule carrying the hereditary materials and biological instructions in humans and all other organisms. Nearly every cell in a person’s body has the same DNA. DNA is found mainly in the nucleus of the cell, and some are found in the mitochondria that generate energy the cell needs to perform all actions. Half of the nuclear DNA is inherited from the male parent and half from the female parent. All the mitochondrial DNA (mtDNA) is inherited from the female parent because only egg cells, and not sperm cells, keep their mitochondria during fertilization.¹

Genome: An organism’s complete set of nuclear DNA is called its genome.

The Structure of DNA:

Nucleotides: Structurally DNA is made of chemical building blocks called nucleotides which have three parts: a phosphate group, a sugar group and one of four types of nitrogen bases which are- adenine (A), thymine (T), guanine (G) and (C) cytosine. The nucleotides link together with alternating phosphate and sugar groups with the base always attached to the sugar. The bases always pair up with each other by hydrogen bonds- A with T and C with G to form units of base pairs. A DNA consists of almost 3.3 billion bases/nucleotides pairs ² and almost 99 percent of those bases are the same in all people. Just like a word must contain an order of specific alphabets to be meaningful, there are orders or sequences of these bases that contain information about which protein to make or what cell regulatory activity is needed.³ For example, the sequence ATCGTT might instruct for blue eyes, while ATCGCT might instruct for brown.¹ The nucleotide links are arranged in two long strands that form a spiral structure called double helix. This double helix resembles a ladder where the base pairs form the rung of the ladder, and the side phosphate and sugar molecules form the vertical sidebars of the ladder.³

Chromatin: 150 base pairs of DNA sequence first wraps around a complex protein called Histone to form a bead like structure called nucleosome. Nucleosomes “beads” are strung along the DNA strand which condenses to form chromatin.⁴

The chromatins are of two types- Euchromatin and Heterochromatin. When cells are doing normal activities, the chromatin is dispersed in the nucleus and euchromatin gets exposed to transcribe to make mRNAs which translate to form proteins outside the nucleus. The heterochromatins are thought to be inactive and are located more along the centromere and telomere parts of the chromosomes.⁵

DNA unwinds when it is replicating itself and also when proteins need to be made for biological functions. Each strand of the double helix divides from the middle to become the template for duplicating the sequence of the bases.³ When cells enter division phase, all chromatin strands first make copies of themselves through this DNA replication process. Then the chromatins undergo massive compression to form the chromosomes right before cell division. Eventually when cells divide, chromosomes separate, and each cell inherits copies of the same number of chromatins of parent cell.⁵

Chromosomes:  DNA is tightly packed into chromosomes to fit into the cell nucleus and is actually six feet long when uncoiled. The long DNA is divided into 23 pairs or 46 chromosomes in each cell. Chromosomes become visible only during cell division and DNA takes its compact chromosome form during cell division to enable transfer to new cells.¹

Out of the 23 pairs, 22 are numbered autosomes that look the same in both sexes and one is a pair of sex chromosome where females have XX and males have XY chromosome. Each pair inherit one chromosome from each parent. Sizes of chromosomes differ, the X chromosome is three times the size of the Y chromosome and contains 900 genes, compared to the 55 in Y .⁶

Structurally each chromosome has a centromere that divides the chromosome into a short “p arm” and a longer “q arm”. The location of the centromere on each chromosome gives it the shape and helps describe the location of genes.

The Function of DNA:

Genes: Proteins give the human body its whole structure and genes are part of the genome that encodes information to make those proteins. Functionally the DNA has protein coding parts and non-coding parts. Genes are protein coding parts of DNA that contain instructions to make a protein or segments of protein that carry out different functions of cells. Genes are the functional units carrying information. The size of a gene may vary greatly, ranging from about 1,000 bases to 1 million bases in humans. A human genome has approximately 20,000 genes.¹ Protein coding genes only make up about one to two percent of the DNA where DNA sequences are converted to messages to form proteins for bodily functions. The rest 98 to 99 percent of DNA are non-coding DNA involved in regulating the gene activities- like signals to make the transfer RNAs, ribosomal RNAs, microRNAs, form the end telomeres that protect ends of chromosomes from being degraded, etc. Non-coding DNA passes information in other subtle ways than making proteins.^8,2,9,1

Gene Regulation & Gene Expression:

Almost all human cells contain the same set of DNA instructions, yet the human body is composed of 200 different types of cells.¹¹ Gene regulation controls which genes in the genome are to be turned on/ expressed in response to both internal and external changes. Gene regulation controls the different sets of active genes in different cells to be activated to respond to different functions.¹²

Gene Expression is the most significant part of the entire cell biologic function.  It is usually triggered by intra or extracellular matrix changes where a hormone or enzyme or an antigen binds to cell wall receptor which changes configuration and enters the cell and then binds to DNA promoter site to initiate the transcription process. Transcription is the first step of gene expression where the triggers decode the gene information to make an RNA copy of the gene’s DNA sequence.  RNA polymerase enzyme involved in transcription separates the DNA strand to use the single stranded DNA template of the gene to synthesize the pre-messenger RNA which is identical to the DNA sequence except that instead of Thymine, the RNA contains Uracil.  This RNA undergoes splicing where introns are chopped out and exons are stuck back to form messenger RNAs (mRNAs) or the transcript.

The mRNAs are then translated outside the nucleus to make proteins or peptides for specific uses. Not all genes are transcribed all the time. Instead, transcription is controlled individually for each gene.¹⁴

Genetic Code and Codon:

A specific gene is expressed to make a specific protein. The coded information within the gene that allows DNA and RNA to compile the amino acids to make that protein is known as genetic code.¹⁵

Genetic code is composed of codons. Each codon is a three-nucleotide sequence, and one codon represents a particular amino acid. Genetic code’s unit is codon.¹⁶ Amino acids are the building blocks of proteins and there are 20 amino acids. Amino acids are represented by specific codon. The codons are basically the letters of a word that instructs a specific amino acid to be added to a polypeptide chain to make the protein and each amino acid may have different codons. For instance, the amino acid valine can be represented by four different codons (GUU, GUC, GUA, GUG). There are 64 different codons, 61 of them represent the 20 amino acids and 3 are used as stop signals to end protein production.

The most important discovery to date is that the genetic code is almost universal, all species use the same genetic codes.¹⁵ Although all stages of gene expression can be regulated, the main control point for many genes is transcription.¹⁷

Genetic Mutation and Genetic Disorders:

DNAs have natural protective features in the form of stop codons, the end telomeres protecting the ends of chromosomes from being degraded, and the non-coding parts of the DNA regulating other activities. However, changes in DNA sequence can happen during cell division when the cell is making a copy of itself- this is known as gene mutation. Mutation can be the natural selection to help humans adapt better to their environment or can lead to terminal diseases like cancer. Germline mutations are changes in the genes of eggs or sperms that affects the offspring but all the other gene mutations in other cells are somatic mutation which are not passed down from parents.

Errors in copying the DNA sequence might replace, delete, or insert the bases or codons leading to dysfunctional cells. Not all mutations are harmful. It can be hereditary, linked to the sex chromosomes or it can happen to anyone due to internal or external factors.¹⁹

Changes in DNA, genes, and chromosomes can manifest in genetic disorders. The genetic conditions can be from anyone of the following:

• Single gene mutation (monogenic)
• Multiple gene mutation (multifactorial inheritance)
• Mutation of one or more chromosomes
• Environmental factors- chemical exposure, UV rays, change in genetic makeup due to personal habits or unidentified causes.¹⁹

Some Common Genetic Disorders:

Although disease related genes and genes related new diseases are now being discovered almost every month, there are some very common genetic disorders known to everyone.

Alzheimer’s disease- APP gene on chromosome 21, PSEN1 on chromosome14, and PSEN2 on chromosome 1 are the genetic variants that can cause Alzheimer’s disease.²⁰ Cystic fibrosis- inherited genetic disorder from mutation of CFTR gene.²¹ Down syndrome- a chromosomal disorder where there is an additional chromosome 21.²² Sickle cell disease- mutation of hemoglobin A producing HBB gene inherited from parent/s.²³

Many types of breasts, ovarian, prostatic and pancreatic cancers are due to genetic mutations or over expression of genes.¹⁹

The Therapeutic and Diagnostic Significance of Genome Sequencing:

Genomics is the study of all the genes of a person including single gene and multiple gene disorders, genes’ interaction with each other, and the environment and lifestyle conditioning other than the hereditary factors that cause some of the common diseases like asthma, diabetes, cancer, hypertension. 99.9 percent genetic makeup of all human beings are the same but that 0.1 percent hold clues about different manifestation of diseases among people.

Proteomics is the study of proteins formed in organisms, tissue or cells that can identify abnormal proteins causing diseases which may happen due to over expression of a gene.²⁴

Gene therapy uses genomics and proteomics to enable development of new drugs to treat, prevent or cure diseases, and new way of delivering the drugs for targeted drug and immunotherapy.^25,24

Gene editing is the technology where a single base can be removed or changed, or an entirely new gene can be inserted along the DNA strand. Gene editing can literally rewrite DNA. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) is the most popular technique where Cas9 is an enzyme that acts like a pair of scissors that can cut DNA.²⁶

Different types of cells arise from different gene expressions in our genome. The many steps involved in gene expression result in either protein production or regulation of other cell activities by the noncoding DNAs and in principle, therapeutics can be developed to regulate activities at any level. However, for most genes the initiation of RNA transcription is the most important point of control.²⁷ 

Advanced technologies now can measure mRNA expression of every gene in the entire genome. It has become the most powerful tool to identify which genes are turned on, how much and in which location. Gene expression is also measured by observing a trait, for example the measurement of protein activity that will identify the specific gene. Different trends can also be measured on reading all the genes being active at the same time to produce that trend. So, when the malfunctioning gene can be identified that is either being expressed excessively or is absent then, gene editing can rewrite the DNA restructure and therapeutics can be made to fight the disease.¹⁸

Ultimately, gene sequencing, genomics and proteomics will play the most critical role as part of routine medical screening for prevention and early diagnosis of diseases, cure of disease, designing target therapy with customized medicine, discover the biomarkers in body fluids especially blood and urine to easily diagnose the disease and invent the right route of delivering the therapeutics. A rapidly growing area currently not even at the nascent stage in Bangladesh. Is it not time to change this scenario?