Human Genome

• The human genome comprises approximately 3 billion base pairs of DNA.
• The Human Genome Project enabled the first DNA sequencing of the entire human genome (published in 2001).
• Genome sequence information for humans and other species is freely accessible through portals like the National Center for Biotechnology Information and Ensembl.
• Most DNA resides within the nucleus as chromosomes (nuclear DNA or nuclear genome), with a small amount in the mitochondria (mitochondrial genome).
• The human nuclear genome encodes about 20,000 protein-coding genes, containing both protein-coding (exon) and non-coding (intron) sequences.
• The genome includes almost 22,000 genes encoding RNA molecules, such as rRNA and tRNA, which perform various cellular roles including gene expression regulation.
• The human genome contains over 14,000 pseudogenes, which are imperfect copies of protein-coding genes that can no longer encode proteins.
• Some pseudogenes may regulate their protein-coding relatives; dysregulation of pseudogene-encoded transcripts has been linked to cancer.
• Gene distribution varies between chromosomes, with chromosome 19 being gene-dense and chromosomes 13 and 18 being relatively gene-poor.

Variation vs. Mutation

• Any heritable change in the DNA sequence (in somatic or germ-line cells) is termed a mutation.
• These mutations may or may not lead to observable differences (phenotype).
• Historically, alterations associated with disease were called mutations.
• Modern genetics uses the term "variants" to refer to any differences from the reference sequence, avoiding the negative connotation of "mutation".
• Variants can be classified as benign (not associated with disease) or pathogenic (associated with disease).
• Variants of uncertain significance (VUS) are human DNA variants with unknown effects.
• Alleles refer to two or more different versions of a DNA sequence at a particular location in the genome.
• Minor allele frequency (MAF) is the frequency at which a particular variant occurs in a population, calculated by analyzing many human genomes.

Types of Variants in the Human Genome

• The most frequent variants in the genome involve the substitution of a single base pair, referred to as single nucleotide variants (SNV) or single nucleotide polymorphisms (SNP) based on MAF.
• It is estimated that there are at least 11 million SNPs in the human genome, averaging about 1 per 300 bp.
• A variant can be called a polymorphism when the MAF is at least 1%.
• Insertions or deletions (indels) of less than 1000 bp are relatively common, with the smallest indels occurring most abundantly.
• Variants affecting segments of DNA greater than 1000 bp (1 kb) are referred to as "Structural variants."
• Structural variants include translocations, inversions, large deletions, and copy number variants (CNV).
• CNVs are genome segments ranging from 1000 to millions of bp, with copy numbers varying from zero to several in healthy individuals; CNVs account for approximately 12% of the human genome sequence.
• A copy number polymorphism (CNP) is a CNV with a population frequency of 1% or more.
• Human genomes contain numerous repetitive sequences, including interspersed repeats that constitute roughly 45% of the genome.
• Tandem repeats are repeated units arranged head-to-tail in arrays; variation in the number of repeats generates multiple alleles and can be used in identifying individuals.
• Tandem repeats include mini-satellites and micro-satellites.
• Although generally inherited stably, expansion of repeats in some microsatellites is associated with disease.

Variation Between Healthy Individuals

• Comparison of a random human genome with the reference sequence shows approximately 3 million SNPs and approximately 2000 structural variants.
• Genomes of two unrelated individuals differ by about 0.5% (approximately 15 million bp), mostly attributed to CNVs and large deletions.
• Despite most variation lying within non-coding DNA, each individual has, on average, several hundred predicted variants that are damaging to gene function.

Chromosomal Abnormalities

• Most human cells contain a full diploid genome, consisting of 2 meters of DNA arranged into 46 chromosomes: 22 homologous autosomal pairs and sex chromosomes (2 X chromosomes in females, X and Y in males).
• Exceptions include erythrocytes, platelets, and haploid germ-line cells (sperm and eggs), which contain 23 chromosomes.
• Mechanisms ensuring complete genome inheritance in daughter cells during cell division occasionally make mistakes, leading to chromosomal abnormalities.
• Chromosomal abnormalities are divided into numerical abnormalities, resulting in daughter cells with too many or too few chromosomes, and structural abnormalities.
• The normal chromosome complement of a species is its karyotype, denoted as 46,XX (female) or 46,XY (male) in humans.

Numerical Abnormalities

• Polyploidy is an abnormality in which a cell contains more than two complete sets of the human haploid genome.
• Aneuploidy arises when a gamete contains more or fewer chromosomes than the normal complement resulting from non-disjunction.
• Non-disjunction occurs when replicated chromosomes do not separate properly during cell division, either in meiosis I (non-disjunction of paired chromosomes) or meiosis II (non-disjunction of sister chromatids).
• Most aneuploidies are lethal.
• Foetuses with trisomy 13 or 18 can survive to term, and individuals with trisomy 21 can survive beyond age 40.
• An extra autosome generally causes severe developmental abnormalities, and only trisomies of small, gene-poor chromosomes are typically tolerated.
• Autosomal monosomies have even more severe consequences, invariably leading to miscarriage during early pregnancy.
• Developmental consequences of trisomies and monosomies result from imbalances in critical gene products encoded on the affected chromosomes.
• Major features of Down syndrome (DS) are associated with the presence of three copies of a 1.6-Mb region at chromosome location 21q22.2, called the Down Syndrome Critical Region.

Structural Abnormalities

• DNA damage from radiation or mutagenic chemicals can lead to chromosome breaks.
• Cell cycle check-points prevent cells with unrepaired chromosome breaks.
• DNA repair mechanisms attempt to repair chromosome breaks but can occasionally repair broken chromosomes incorrectly, resulting in structurally abnormal chromosomes.
• Wolf-Hirschhorn syndrome is caused by partial deletion of the short arm of chromosome 4.
• Single-gene disorders are conditions and diseases that depend on the genotype at a single gene, with inheritance passed down according to Mendel's laws.
• These diseases are often called Mendelian.
• Not all inherited disorders follow Mendel's laws (e.g., triplet-repeat diseases and imprinting disorders).
• Mendelian diseases can be recognized by their characteristic inheritance patterns in family trees or pedigrees.
• Pedigrees can reveal if the locus resides on an autosome or sex chromosome and if a genetic variant is dominant or recessive.

Classification of Mendelian Diseases

• The major classes of Mendelian diseases are autosomal and sex-chromosome linked.
• Autosomal diseases are divided into autosomal dominant and autosomal recessive based on inheritance patterns.
• Sex-chromosome linked Mendelian diseases are divided into X-linked dominant, X-linked recessive, and Y-linked.
• A dominant allele leads to a particular phenotype (e.g., a genetic disorder) regardless of whether the second allele is 'normal' or not.
• A recessive allele does not lead to a phenotype on its own; the remaining 'normal' allele is sufficient to compensate.
• A phenotype is displayed when a person inherits recessive alleles of a gene from both parents because no 'normal' allele is present to compensate.
• A person with an autosomal dominant disorder usually has at least one similarly affected parent, and on average, 50% of their children will have the disorder.
• Autosomal dominant disorders occur in each generation, affect both males and females, and can be transmitted from either parent to offspring of either sex; one copy of the 'affected' gene is enough to show related symptoms.
• Autosomal recessive conditions are caused by loss-of-function pathogenic variants that do not lead to a recognizable phenotype; the presence of a second, functional allele of the gene on the homologous autosome is sufficient to compensate.
• Such conditions only manifest themselves in individuals who carry 2 pathogenic copies of the same autosome.
• Individuals with autosomal recessive conditions have two unaffected parents, who are both non-symptomatic, heterozygous carriers of a single pathogenic allele.
• The children of two carrier parents have a 25% chance of inheriting both pathogenic variants.
• For an autosomal recessive disorder to manifest, the child would need both parents to be carriers of the defected gene; only when both copies of the gene are defected would the person suffer.
• With 1 defective copy, the person would be an asymptomatic carrier.

X-Linked Recessive Disorders

• Diseases caused by recessive variants in loci located on the X chromosome affect females and males differently.
• Males have a single X chromosome; therefore if they carry a pathogenic variant, they have no second allele to compensate, and they will be affected by the disease.
• All daughters of affected males will inherit their X chromosome and be carriers, while their sons will be unaffected.
• Females carry two X chromosomes; therefore, they will only be affected by the disease if they inherited 2 copies of pathogenic variants from both of their parents (e.g., Haemophilia A).
• Differences in the manifestation of disease depend on the child's gender.
• A dominant pathogenic variant on the X chromosome will typically affect both males and females.
• In X-linked dominant disorders, all daughters of an affected male will inherit the condition, while all of his sons will be unaffected.
• In the case of an affected female, her children have a 50% chance of being affected if she has a single pathogenic variant.
• If she has inherited pathogenic variants from each of her parents, all of her children will also be affected.
• Rare in comparison to X-linked dominant ones, Y-linked single-gene disorders are especially rare, given the Y chromosome’s size and gene count.
• As much of the Y chromosome exists in a hemizygous state (with the exception of genes with homologues on the X chromosome), recessive and dominant definitions do not apply.
• Affected males also have affected fathers, unless a de novo mutation has occurred, and all their sons will be affected.
• Since daughters of affected males will inherit their father's normal X chromosome and not the affected Y chromosome, they will be unaffected.
• Non-obstructive spermatogenic failure, which leads to fertility problems in males, is an example of a Y-linked condition.
• Mitochondria are cellular organelles containing a genome which is independent of the nuclear genome.
• The mitochondrial genome exists as a circular, dsDNA molecule of approximately 16500 bp encoding 37 genes, each of which is vital to the function of mitochondria.
• Numerous nuclear DNA encoded proteins also contribute to the formation and function of the mitochondria.
• Each cell contains thousands of mitochondria, and each mitochondrion contains multiple copies of the mitochondrial genome.
• 13 of the 37 mitochondrial genes encode subunits of the 4 respiratory complexes involved in oxidative phosphorylation, situated in the inner membrane, while the rest code for 22 tRNAs and 2rRNAs.
• Almost 93% of mtDNA is coding, compared with the nuclear genome of which only 3% is coding, and mtDNA is free from introns, histones and epigenetic marks.
• MtDNA is subject to a mutation rate 100-times higher than that of the nuclear genome, since the mtDNA repair systems are more error prone than nuclear DNA repair systems and because the internal environment of the organelle has more reactive molecules that can damage DNA (from the products of the respiratory transport chain).
• A female will pass mtDNA is only inherited maternally - on to all her children while a male will not normally pass on any of his mitochondria.
• Heteroplasmy is an important feature of the mitochondrial genome this means that not all copies of the mitochondrial genome within a cell are the same.
• The cell is said to be homoplasmic for the mutation if a particular variant is present in all copies of the mitochondrial genome in a cell.
• If some mitochondria contain the mutation, and others which do not are referred to as heteroplasmy.
• An offspring may inherit a high or low proportion of mutant mitochondria from a carrier mother.
• Many conditions are associated with mitochondria from age-related hearing loss to specific forms of epilepsy and diabetes ; genome mutations.
• A mitochondrially inherited disorder of vision, with reported incidence of approximately 1/30000 to 1/50000 births in European populations Leber hereditary optic neuropathy (LHON).
• Mutations in a number of mitochondrial genes, are known to cause LHON, including those encoding several NADH dehydrogenases (MT-ND1,4 and 6).
• Genetic diagnosis of the mutations causing mitochondrial disease such as LHON can be carried out using PCR-based techniques.
• Epigenetics and change in DNA sequencing may lead to disease states, but there is also an additional level of complex information needed for a human to be healthy.
• The study of heritable changes in gene expression without a change in DNA sequence defines epigenetics (i.e., changing the phenotype without changing the genotype).
• There is another level of genetic control in addition to a person’s DNA sequence which determines which genes are turned off and which are turned on.

Epigenetic Modification

• The two main types of epigenetic modification are DNA methylation and Histone modification.

DNA Methylation

• This reaction adds a methyl group to DNA and catalysed by enzymes known as DNA methyltransferases; it occurs on 5-position (C5) of cytosine nucleotides that are found next to a guanine nucleotide in the DNA sequence that are linked by a phosphate group to form a CpG dinucleotide and forms 5-methyl-cytosine.

Histone Modifications

• There are multiple types of histone modifications which are catalyzed by a number of enzyme families; the most well characterized modifications are acetylation and methylation of histones H3 and H4; the modifications directly alter the DNA-protein interactions to change how chromatin is structured which will alter the ability for a gene to be transcribed and expressed.
• Changes in epigenetic level are often related to multifactorial disorders such as cancer or diabetes.

Multifactorial Disorders

• Diabetes, heart diseases and schizophrenia, are the outcome of a complex interplay of multiple genetic and environmental influences.
• The impact of an individual variant in one gene may be very small, but when present together with multiple variants in other genes, in the context of a particular environment, this may lead to an increased risk of disease.
• Type 2 diabetes (T2D) is such complex disorders and is increasing in incidence across the world.
• Both genetic factors and environmental factors (lack of physical exercise, high-calorie food intake, family history, stress) contribute to the manifestation of diabetes.
• Epigenetics also plays a significant role in complex diseases like T2D.
• Lifestyle choices and environmental exposures also lead to epigenetic change.
• Tobacco smoking leads to decreased methylation in several genes associated with T2D, including KCNQ1, and exercise has been shown to promote methylation changes in T2D- associated genes as well as altering histone deacetylase expression.
• Because they contribute to diabetes, such kind of epigenetic changes are also happening in other medical conditions.

Cancer

• The genome of a cancer cell is full of mutations.
• Some cancers such as lung cancer, there may be hundreds of thousands of mutations.
• In cancer cells such mutations are observable at the level of the karyotype, including whole and part chromosome duplications, deletions, inversions and translocations.
• Cancer cells typically carry large numbers of point-mutations.
• Oncogenes are genes whose activation contributes to the development of cancer.
• Oncogenes are usually mutated versions or pathogenic variants of normal cellular genes. H-RAS, K-RAS these are increasingly expressed in different cancer types, so they are oncogenes.
• The unmutated genes are often called proto-oncogenes.
• Tumor suppressor genes (TSG) are genes whose action inhibits the growth of tumor cells.
• Their inactivation is advantageous to a cancer cell.
• Consequently, the function of several TSGs is lost from all forms of cancer.
• Loss can occur due to deletion mutation or by epigenetic changes.
• P53, p21 these are known tumor suppressor genes.