Genomics Primer

Introduction

The Genomics Primer serves as a foundation for practitioners to reference for basic definitions and concepts in the field of genomic medicine. Upon successful completion of this module, participants should be better able to:

• Identify the components of a eukaryotic cell.
• Understand the composition and structure of DNA.
• Identify the three components used to describe a gene’s location on a chromosome.
• Identify the steps of the gene expression process.
• Identify the types of chromosome abnormalities.

What are Eukaryotic Cells?

A cell is the basic building block of human life. The human body consists of trillions of cells. Human cells are comprised of many parts, each of which have specific functions. The cytoskeleton consists of a network of fibers that helps to retain a cell’s shape and allows the cell to move. The cytoskeleton also helps guide the movement of the organelles, which are structures within the cell that execute certain functions. Cytoplasm, a jelly-like fluid, surrounds the organelles and the cell’s nucleus, which is a structure that contains deoxyribonucleic acid (DNA). Each organelle has a certain job to perform within the cell. Types of organelles include:

• Endoplasmic reticulum – assists with sorting, processing and transportation of protein and lipid molecules. It is the largest organelle.
• Golgi apparatus – bundles molecules (such as proteins and lipids) processed by the endoplasmic reticulum and transports them out of the cell.
• Mitochondrion – converts food into energy that the cell can use and synthesizes adenosine triphosphate by a process called oxidative phosphorylation. Mitochondria are the powerhouse of the cell.
• Ribosome – creates proteins for use inside and outside the cell.
• Lysosome and peroxisome – digest foreign bacteria in the cell, rid the cell of toxic materials and recycle worn out cell components. These organelles act as the recycling center of the cell.

The nucleus is often referred to as the control center, or brain, of the cell and contains the DNA, or genetic material. The nucleus is surrounded by the nuclear envelope.

Cells that contain these features (i.e., cytoskeleton, organelles surrounded by cytoplasm and nucleus surrounded by nuclear envelope) are called eukaryotic cells. Human cells are eukaryotic cells.

DNA Definition

What is DNA?

Deoxyribonucleic acid (DNA) is the cell’s hereditary material and contains instructions for development, growth and reproduction. DNA is passed from generation to generation in humans and many other organisms. The same DNA is located in nearly every cell of the human body. DNA is mostly located within chromosomes in the nucleus, but some DNA is also found in the mitochondria. Chromosomes consist of DNA coiled around histones (alkaline proteins). If extended, the DNA would measure approximately 2 meters.

DNA is made up of four chemical (nitrogen) bases:

Purines

• Adenine
• Guanine

Pyrimidines

• Cytosine
• Thymine

The side-by-side arrangement of bases in a particular sequence, unique to each human (or other organism), spells out the exact instructions needed to create that person and also gives the person his or her unique phenotype or traits (distinguishing characteristics).

Genes, the basic physical and functional units of heredity, are composed of DNA sequences. Genes serve as the blueprint for the production of proteins. Only about 2% of the entire human genome (complete set of genetic information for a person) consists of genes.

DNA Composition and Structure

• Adenine + Thymine
• Cytosine + Guanine

These pairs are called base pairs. The base pairs are held together by a hydrogen bond. One base plus a deoxyribose sugar molecule and a phosphate group create a nucleotide.

Nucleotides are arranged in two long strands and are held together by the sugar-phosphate backbone. The nucleotide strands form a spiral double helix that looks similar to a ladder. The sides of the ladder consist of sugar and phosphate molecules. The middle rungs are formed by the base pairs (and the hydrogen bond). Nucleotides are the building blocks of nucleic acids.

Types of DNA in the Cell

There are two types of DNA in the cell – autosomal DNA and mitochondrial DNA. Autosomal DNA (also called nuclear DNA) is packaged into 22 paired chromosomes. In each pair of autosomes, one was inherited from the mother, and one was inherited from the father. Autosomal DNA is passed down from both the mother and the father and provides clues to a person’s ancestry.

As noted in the section “What are Eukaryotic Cells?”, mitochondria are organelles that are responsible for the cell’s energy production. Mitochondria contain their own DNA called mitochondrial DNA. Mitochondrial DNA has one chromosome that codes for the specific proteins needed for the metabolic processes that mitochondria perform. Mitochondrial DNA replicates separately from the rest of the cell and is passed down only from the mother.

Noncoding and Coding DNA

Noncoding DNA is sometimes referred to as junk DNA. However, noncoding DNA does have a purpose. Noncoding DNA typically refers to any DNA that does not code for a protein. This type of DNA is now referred to as regulatory DNA. The regulatory function of this DNA is to determine when and where some genes are transcribed. Noncoding DNA also provides chromosomal structure and binding sites for regulatory proteins. Much research is being conducted on noncoding DNA.

Coding DNA is responsible for harboring the specific DNA sequences that encode instructions for making proteins.

What are Chromosomes?

Chromosomes are thread-like structures in which DNA is tightly packaged within the nucleus. DNA is coiled around proteins called histones, which provide the structural support. Chromosomes help ensure that DNA is replicated and distributed appropriately during cell division. Each chromosome has a centromere, which divides the chromosome into two sections – the p (short) arm and the q (long) arm. The centromere is located at the cell’s constriction point, which may or may not be the center of the chromosome.

At the end of each chromosome is a repetitive nucleotide sequence cap called a telomere. In vertebrates, the telomere is a TTAGGG sequence repeated to approximately 15,000 base pairs. These DNA regions serve a critical role of preserving the genomic sequence by protecting the genome from degradation, and inhibiting chromosomal fusion and recombination. These regions are also involved in chromosome organization within the nucleus.

In humans, 46 chromosomes are arranged in 23 pairs, including 22 pairs of chromosomes called autosomes. Autosomes are labeled 1-22 for reference. Each chromosome pair consists of one chromosome inherited from the mother and one from the father.

In addition to the 22 numbered autosomes, humans also have one pair of sex chromosomes called an allosome. Instead of labeling these chromosome pairs with numbers, allosomes are labeled with letters such as XX and XY. Females have two copies of the X chromosome (one inherited from the mother and one from the father). Males have one copy of the X chromosome (inherited from the mother) and one copy of the Y chromosome (inherited from the father).

Arranged on the chromosomes are genes. Genes are made of DNA and contain the instructions for building proteins and are integral in making and maintaining the human body.

Locating a Gene

The cytogenetic location, a standardized way of describing a gene’s location on the chromosome, consists of a combination of numbers and letters and is made up of three components:

• Number or letter of the chromosome (1-23, X or Y)
• Arm of the chromosome (p or q)
• Position of the gene on the arm (cytogenetic bands). The position is dependent on the light and dark bands that appear on the chromosome when stained and is expressed as a two-digit number (one digit represents region and one represents band). Sometimes the digits are followed by a decimal point and one or more digits. These additional digits represent the distance from the centromere (increasing numeric value indicates farther distance from centromere). “Cen”, “ter”, and “tel” are also used to describe the position of the gene on the arm.

Cen – close to the centromere
Ter (terminus) – close to end of either the p or q arms
Tel (telomere) – close to end of either the p or q arms

Example
Gene: Anaplastic lymphoma kinase receptor
Chromosomal location: 2p23
Location description: chromosome 2, p arm, position 23

Chromosome vs. Molecular Locations

Chromosome location, or cytogenetic location, is one way to describe the location of gene on a chromosome. Another way to identify the location of a gene is by using the molecular location. The sequencing of the base pairs describes the molecular location of the gene on a chromosome. The molecular location is more precise; however, small variations in the address may occur between research groups as a result of varying genome sequencing methods.

For example, in humans, the chromosomal location of the EGFR gene is 7p12, while the molecular location is Chromosome 7, NC_000007.14 (base pairs 55,019,032 to 55,207,338).

Mitosis vs. Meiosis

Cells divide through two processes: mitosis and meiosis. In both processes, diploid cells (containing two sets of chromosomes, or 46 chromosomes) divide. In mitosis, the diploid “parent” cell divides and produces two diploid “daughter” cells. However, in meiosis, the parent cell produces four haploid daughter cells (each containing half of the parent cells chromosomes, or 23 chromosomes).

The critical difference between mitosis and meiosis is that mitosis produces two genetically identical daughter cells, whereas meiosis produces four genetically different daughter cells.

The phases of cell division are similar for both mitosis and meiosis, and both processes result in cytokinesis (cytoplasmic division of the daughter cells). However, in meiosis, the cycle occurs twice (meiosis I and meiosis II) before the four haploid daughter cells are produced.

Another difference between the stages of mitosis and meiosis is that in meiosis, homologous chromosomes pair up during metaphase instead of chromatids. In a homologous pair, one chromosome comes from the mother, and one chromosome comes from the father. Homologous chromosomes are very similar, but they are not identical. They carry the same genes (e.g., hair or eye color), but they may not code for the same trait (e.g., blonde hair or brown eyes).

Stages of Cell Division

Interphase – Replication of DNA. Most of a cell’s time is spent in interphase. Occurs before cell division. Consists of three stages: Gap 1 (growth), S phase (DNA replication) and Gap 2 (continues growth, prepares for cell division).

Prophase – Chromosomes condense. Mitotic spindle forms.

Metaphase – Chromosomes line up in the middle of the cell.

Anaphase – Chromatids separate from one another and are pull toward opposite sides of the cell.

Telophase – Division of the cell contents into two new cells occurs (cytokinesis).

What is a Genome?

A genome is an organism’s complete set of genetic information. A genome includes all of the hereditary instructions for creating and maintaining life, as well as instructions for reproduction. The human genome, like all other cellular life forms, consists of DNA and includes both the nuclear and mitochondrial DNA. This is in contrast to ribonucleuic acid (RNA) viruses, whose genome is comprised of RNA.

In 1990, an international research effort known as the Human Genome Project was undertaken to determine the sequence of the human genome and identify the genes it contains. The Project also sequenced the genomes of several additional organisms important to medical research, including the mouse and the fruit fly.

Human Genome Project

The Human Genome Project (HGP) was an international, collaborative research effort to determine the sequence of the human genome and identify the genes that it contains. The HGP formally began in 1990 and was completed in 2003. The initial goals of the HGP were to develop technology to increase efficiency and lower the cost of DNA sequencing, analyze the structure of human DNA and provide a complete and accurate sequence of the 3 billion DNA base pairs that make up the human genome. At the start of the HGP, 50,000 to 140,000 genes were estimated to make up the human genome. It is now known that the human genome contains roughly 3 billion base pairs and 20,500 genes. The full human sequence was published in 2003 (10) (with the exception of 1% due to limitations in current technology). The HGP has contributed to the identification of more than 1,800 disease genes.

Regulation of Gene Expression in Eukaryotes

What is RNA?

Ribonucleic acid (RNA) is another molecule present in a cell. RNA is located in the cell nucleus and is composed of a single strand of alternating sugar (ribose) and phosphate groups, along with nitrogen bases (adenine, uracil, cytosine, guanine). Thymine (found in DNA) is not found in RNA; it is replaced by uracil. For RNA, adenine pairs with uracil, while guanine pairs with cytosine.

The main function of RNA is to produce proteins using a process called protein synthesis, which consists of two phases, transcription and translation.

Gene Expression

Gene expression is the phenotypic manifestation of genes by the processes of transcription and translation. Gene expression via transcription and translation is a fundamental principle of molecular biology that is often referred to as the central dogma of molecular biology.

Gene expression in humans is complex and highly regulated. Regulation occurs at many points during the transcription and translation processes and involves epigenomic compounds, which are chemical compounds and proteins that can attach to DNA and influence gene expression.

Transcription

Transcription occurs in the cell’s nucleus. The main purpose of the transcription process is to produce and process messenger RNA (mRNA). RNA is involved in coding, decoding, regulation and expression of genes. RNA is single stranded and contains the nucleotide uracil instead of thymine. RNA also contains ribose sugar molecules. The mRNA contains the information for making a protein and transports the information out of the nucleus and into the cell’s cytoplasm.

Translation

Translation occurs in the cell’s cytoplasm. The main purpose of the translation process is protein synthesis. Ribosomes reach the mRNA and read the sequence of the bases. Each sequence of three bases is called a codon, and each codon contains the instructions for one amino acid. Transfer RNA (tRNA) is another type of RNA. The tRNA assembles the protein using the amino acids. The protein continues to be built until a stop codon is encountered. A stop codon is a three-base sequence that does not code an amino acid.

Three different types of RNA molecules are required for transcription and translation, and each type of RNA has a different function. When genes are “turned on,” RNA polymerase attaches to the start of the gene and then moves along DNA, creating a single strand of mRNA. mRNA contains the protein coding instructions and moves from the cell nucleus through the cytoplasm. In the mRNA, each “triplet” (or three nucleotide sequence) forms a codon. Codons on the mRNA are read by the ribosomal RNA (rRNA; a component of the ribosome) and matched up to the transfer RNA (tRNA) molecule. Each codon specifies a particular amino acid (the building blocks of a protein).

When the ribosome attaches to the mRNA, the codons are read. tRNA matches up to each codon delivering the matching amino acid and adding to the growing amino acid chain (protein). One start codon (AUG) and several stop codons (UAA, UAG, UGA) indicate the start and stop of the amino acid chain. The start and stop codons do not code for any of the 20 amino acids.

What is a Protein?

Proteins are the result of DNA transcription and translation. Proteins are macromolecules made of one or more polypeptide chains, which are made up of a sequence of 20 different amino acids. Proteins bind to other molecules called ligands. After polypeptide chain(s) are completed, the chain(s) fold over onto themselves to create a 3-dimensional structure. The resulting polypeptide chains direct the function of the protein in the cell.

Proteins facilitate many functions that support human life.

Antibodies bind to foreign particles (e.g., viruses, bacteria) to protect the body. White blood cells, specifically B lymphocytes, produce antibodies. Antibodies are typically “Y” shaped.

Enzymes perform or catalyze chemical reactions in cells (e.g., muscle contraction). Enzymes assist with bodily functions such as digestion and DNA replication.

Messenger proteins transmit signals to coordinate biological processes that occur between different cells, tissues and organs. Hormones (e.g., insulin, oxytocin) are great examples of messenger proteins.

Structural proteins provide structure and support for cells. Actin filaments and microtubules are examples of structural proteins. There are three types of structural proteins: fibrous, globular and membrane. Fibrous proteins form hair, nails and skin.

Transport proteins bind and carry atoms and small molecules. Hemoglobin is a transport protein in red blood cells that is used to carry oxygen from the lungs to other tissues.

Because proteins are critical to supporting human life, it is imperative that they function properly. Dysfunctional proteins can result in uncontrolled tumor growth and spread in patients with cancer.

Gene Regulation

Gene regulation is the process of turning genes on or off. Gene regulation can occur at any point of the transcription-translation process but most often occurs at the transcription level.

Proteins that can be activated by other cells and signals from the environment are called transcription factors. Transcription factors bind to regulatory regions of the gene and increase or decrease the level of transcription. Other mechanisms of gene regulation include regulating the processing of RNA, the stability of mRNA and the rate of translation.

Turning the correct genes on and off is an essential component to maintaining a cell’s functionality.

Epigenetic Modifications

An epigenetic change is a modification to DNA that occurs when a chemical compound or protein attaches to a gene and alters gene expression. The actual DNA sequence is not changed, but rather the chemical or protein is attached to the DNA. Epigenetic changes can be passed down through inheritance or can occur through exposure to environmental substances, as a result of lifestyle behaviors or due to increasing age.

One example of epigenetic change is methylation. Methylation occurs when small molecule methyl groups are added to DNA. The addition of these groups to DNA results in the gene being turned off, and thus the protein made from that gene is not produced.

The epigenome changes throughout a person’s life.

Gene, Genome Editing and CRISPR

Many scientists have contributed to the development of genome-editing technology. Emmanuelle Charpentier, PhD, and Jennifer Douda, PhD, are often considered pioneers in the field. In 2015, they published a paper on using a bacterial system called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) or CRISPR-Cas9 technology to edit genomes.

The CRISPR technology can make precise changes in human DNA by slicing out the incorrect portion of the gene and replacing it. It is a complicated process, but simply put, “guide” RNA and a bacterial enzyme, called Cas-9, bind to and cut DNA. A repair template with the desired change is inserted where the DNA has been cut. Multiple DNA edits can be made simultaneously.

Editing DNA with CRISPR has many advantages. For example, genome editing could potentially prevent or treat genetic diseases such as cystic fibrosis, hemophilia and sickle cell anemia. Research is also being done on DNA editing in the treatment of more complex diseases, such as cancer. CRISPR technology is quick and fairly easy for trained scientists.

Although there are many benefits of using CRISPR, the technology also has some limitations. Although CRISPR technology is precise, it is not perfect. It sometimes cuts DNA that is similar to the guide RNA, but not exact.

CRISPR has been one of the biggest scientific achievements of the century. However, with progress comes considerations. There are complicating ethical issues to evaluate when considering DNA editing. For example, is it appropriate to edit the genomes of human embryos? Should we cure disease? Do edits we make today have unforeseen impacts to future generations? How does commercialization of gene editing technologies fit in? Should CRISPR technology be available to the scientific masses, or should its use be limited to selected experts? These questions remain up for debate as conversations about CRISPR technology continue. While this debate continues, leaders in genetics and bioethics have proposed a moratorium on germline gene editing.

Genetic Alterations

Genetic alternations include chromosomal abnormalities and gene mutations. Chromosomal abnormalities generally arise during cell division. They can be numeric, involving the number of chromosomes, or structural, involving the atypical configuration of one or more chromosomes. Many different chromosome abnormalities have been identified, some of which are associated with genetic disorders and diseases like cancer.

Gene mutations are permanent changes in DNA gene sequence. They can arise during normal DNA replication or in response to environmental factors. There are many classes of gene mutations. Certain mutations cause disease.

Chromosomal Abnormalities

Each human has 46 chromosomes (23 pairs). If a human does not have 46 chromosomes, a chromosomal abnormality has occurred. An umbrella term for a gain or loss of chromosome is aneuploidy.

Chromosome abnormalities often occur during cell division (meiosis and mitosis). There are two main groups of chromosome abnormalities — numeric and structural. Numeric abnormalities, as the name suggests, involve the number of chromosomes. Monosomy occurs when one of the two chromosomes is missing from a pair. An example of a monosomy disorder is Turner syndrome, in which part or all of a female’s second X chromosome is missing. Trisomy occurs in individuals with an extra chromosome. For example, those with Down syndrome have three copies of chromosome 21 instead of two copies.

In addition to chromosome losses or gains, chromosomes can simply be altered, which is known as structural abnormality. Many structural abnormalities exist. A translocation occurs when a piece of one chromosome breaks off and attaches to another chromosome. Deletions occur when a portion of the chromosome breaks and genetic material is lost or deleted. A duplication happens when part of a chromosome is copied and additional genetic material is present. When a chromosome has broken, rotated and reattached, an inversion has occurred. A pericentric inversion occurs in the centromere, and a paracentric inversion occurs in the p or q arms. Isochromosomes are another type structural abnormality in which the chromosome has two identical arms (e.g., two p arms). A dicentric chromosome is a chromosome with two centromeres, and a ring chromosome is one in which the chromosome breaks in two places and the ends fuse together to form a ring shape.

Mutations

A gene mutation is a permanent change in the DNA sequence of a gene. Mutations can occur in a single base pair or in a large segment of a chromosome and even span multiple genes. Mutations can result from endogenous (occurring during DNA replication) or exogenous (environmental) factors. There are two main categories of mutations: germline and somatic.

Germline (hereditary) mutations
Germline mutations are inherited from a parent (i.e., mutation was present in the parent’s egg or sperm cells). A person with a germline mutation will have the mutation in every cell in the body. Germline mutations are the cause of some diseases, such as cystic fibrosis and cancer (e.g., breast and ovarian cancer, melanoma).

Cystic fibrosis is a hereditary genetic disorder that results in a thick, sticky buildup of mucus in the lungs, pancreas and other organs. Cystic fibrosis is the most common genetic disease and arises from a mutation in a single gene named the cystic fibrosis transmembrane regulator gene (CFTR). The location of this gene is on the long arm (q) of chromosome 7 (position 31.2).

Some forms of breast cancer can be hereditary. Two genes are associated with hereditary breast cancer, BRCA1 and BRCA2. The BRCA1 gene is located on chromosome 17, and the BRCA2 gene is located on chromosome 13. Carriers of mutated BRCA1 and BRCA1 genes are at an increased risk for both breast and ovarian cancers.

In approximately 10% of patients with melanoma, hereditary mutations may play a role. On chromosome 9, the gene CDK2N instructs protein development. Proteins made by CDK2N include p16 and p14. These proteins prevent cells from growing uncontrollably and are referred to as tumor suppressors. Some studies have also indicated that genes on chromosomes 1 and 2 may play a role in hereditary melanoma.

Somatic (acquired) mutations
Somatic mutations can occur at any point in a person’s life. These mutations are often caused by environmental or lifestyle factors and can also result from mistakes during cell division. This type of mutation is not passed down from parents to children and thus, is not present in every cell in the body.

Although several types of hereditary cancers can be linked to germline mutations in genes that alter the gene’s original function (eg, tumor suppression), most cancers arise from somatic mutations. Somatic mutations arise after conception and can affect any of the body’s cells, except for germ cells. Approximately 10% of cancers demonstrate both germline and somatic mutations. Alterations in genes, whether they occur in a germline or somatic fashion, change the function of the gene, which may contribute to the development or spread of cancer.

Types of mutations
There are many classes or types of mutations. See table 1-1.

Single nucleotide polymorphisms
A single nucleotide polymorphism (SNP, pronounced snip) is one difference in a single base pair, or nucleotide, in a section of DNA. SNPs result in genetic variation in humans. SNPs can occur with a gene or near a gene, but they are most commonly found in the DNA between genes. To be designated as a SNP, the change in the base pair must be found in at least 1% of the population.

SNPs are common and normal variations in the DNA and are responsible for many of the normal differences between people such as eye color, hair color and blood type. Many SNPs have no negative effects on a person’s health, but some variations may influence the risk of developing certain health problems such as diabetes, heart disease or cancer.

On average, SNPs occur once in every 300 nucleotide base pairs, which means that the human genome has roughly 10 million SNPs.