Human Genome Variation

• Human genome variation can involve alterations in the DNA sequence and chromosomal aberrations.
• Associate Professor Zanda Daneberga presents information on human genome variation.

Terms

• Locus refers to the chromosome location of a specific gene; its plural form is loci.
• Allele: Different or alternative forms of a particular gene or DNA sequence.
• Homozygote: An individual having two identical alleles at a specific locus.
• Heterozygote: Describes an individual possessing two distinct alleles at a particular locus.
• Hemizygote: An individual with unpaired genes in an otherwise diploid cell, such as X-linked genes in males.
• Wild-type: Denotes a single prevailing allele present in more than half the population, is the most common allele. The old way to indicate normal, using the term "normal" is discouraged.
• Variant: Any allele version differing from the wild-type resulting from permanent changes in nucleotide sequence and/or arrangemen.

Changes in nomenclature based on HGVS

• Old term "mutation" signifies an alteration in the DNA sequence, encompasses both general changes and disease-causing changes.
• New terms include "sequence variant", "allelic variant", or "alteration" to avoid confusion with the term "mutation".
• Old term "single nucleotide polymorphism" (SNP) refers to variations at a single nucleotide, could refer to a sequence variation that is not disease-causing or a variant found at a frequency of 1% or higher in a population.
• New terms include "sequence variant", "allelic variant", etc to avoid the many potential meanings of "single nucleotide polymorphism".

The Nature of Genetic Variation

• Recognizing DNA sequence changes necessitates a reference genome, a recognized "gold standard".
• The Human Genome Project completed it's work in 2003 with the original draft sequence of the human genome was released in 2001 (hg7).
• Human Genome Assembly is updated by the Genome Reference Consortium.
• The latest release is GRCh38 (hg38) as of December 2013, with a later update in February 2019.

Genetic Variation

• All genetic variation originates from mutation, which leads to alteration in the human genome.
• Human genome alteration happens at different level.
• Mutation can affect somatic (cancer development) and germline cells (can be passed to the next generation).
• Chromosomal aberrations involve numerical and structural changes.
• Alterations can also occur in the DNA sequence itself.

Chromosomal Aberrations vs. DNA Alterations

• Chromosomal aberrations cause changes in the number and structure of chromosomes.
• DNA alterations occur in the DNA base sequence.
• Chromosomal aberrations can include multiple gene alterations.
• DNA alterations commonly refer to a single-gene alteration or alteration of the non-coding sequence.
• Damages from chromosomal aberration are large scale.
• Damages to nucleotides are small scale compared to chromosomal aberration.

Origin of Genetic Variation

• Spontaneous mutations include errors in DNA replication, the most common source, prevented by DNA polymerase "proofreading" activity.
• Small numbers of extra nucleotides can be inserted into the synthesized polynucleotide, or some nucleotides in the template may not be copied.
• Repeated units of DNA sequence may result in replication slippage.
• Errors in DNA reparation may also occur.
• Errors in recombination may occur during cell division.
• Errors in cell division can occur during meiosis I.
• Induced process of mutation is made by the environment.
• Physical: Ionized radiation and UV are examples of mutation inducing factors.
• Radiation can cause UV-induced dimerization usually resulting in deletion of when the modified strand is copied.
• Chemicals such as oxidative stress, aromatic amines, and deaminating agents can induce point mutations.
• Biological factors like transposons and viruses can induce mutation.
• Average human mutation rate in germ cells is in the range of 1.1-1.7 x 10^-8 per nucleotide site per generation for base-substitution mutations alone.
• The mutation rate to small insertion/deletions is approximately 8% of the base-substitution rate.
• Large structural changes arise at a rate of approximately 0.08% per haploid genome per generation.

Alteration of DNA Sequence: HGVS Molecular Mechanisms

• Allelic Variant (Previously SNP) are generally 2 alleles.
• Molecular mechanism can be 1bp Substitution of one nucleotide in the coding or non-coding regions of the genome, which may or may not alter the amino acid sequence.
• Allelic Variant (Previously Indels) are generally 2 alleles.
• Molecular mechanism can be 1bp - 100bp Insertion or deletion of genome fragment.
• Allelic Variant (Previously STR) are multiple usually -5 or more alleles.
• Molecular mechanism can cause Microsatellites - dinucleotide, trinucleotide, or tetranucletide tandem repeats that are often used in identity or paternity/maternity testing.
• Copy number variation (CVN) is generally 2 or multiple alleles.
• Molecular mechanism can cause Absence or presence of large segments (200bp-1.5Mb) that may involve coding and non-coding regions.
• Mobile element insertions are generally 2 alleles.
• Molecular mechanism can cause Retrotransposition of mobile genetic elements often found in all human chromosomes, such as the LINEs and Alu family.

Type of Allelic Variants and Consequences

• Base substitutions involve:
• Missense mutations: Single nucleotide substitutions in coding regions that alter the genetic code by nonsynonymous replacement of one amino acid.
• Silent mutations: Single nucleotide substitutions in coding regions that alter the genetic code by synonymous replacement of one amino acid, with no change in the amino acid sequence.
• Nonsense mutations: Single nucleotide substitutions that produce one of the three stop codons (UAA, UAG, or UGA) in the messenger RNA (mRNA).
• RNA transcription, processing and translation are affected by:
• Splice site mutations, which occur at exon-intron or intron-exon boundaries, altering the splicing signal needed for proper intron excision.
• Deletions: Loss of one or more base pairs.
• Insertions: Insertion of one or more base pairs.
• Deletions and insertions are especially harmful when the length of the missing or extra base pairs is not a multiple of three.
• Frameshift mutation: It may lead to the shift of reading frame and alter all of the downstream/upstream codons resulting in this altered form of mutation.
• Insertions of mobile elements include insertions of LINE and SINE (Alu) repeats that can cause frameshift mutations.
• Dynamic mutation involves amplification of a simple nucleotide repeat sequence (e.g [CGG]n, [CAG]n), expanding during gametogenesis.

Consequences of Allelic Variants

• Gain-of-function mutations: lead to new protein product (with new function) or overexpression of product or inappropriate expression (wrong time, wrong place etc.).
• Loss-of-function mutations: lead to gene product activity loss.
• Dominant negative mutations: the abnormal protein product interferes with the normal protein product and inhibits its function.

Interpretation of DNA Sequence Alteration

• Pathogenic variants are disease causing.
• Likely pathogenic variants are probably disease causing.
• Benign variants are not disease causing.
• Likely benign variants are probably not disease causing.
• Uncertain significance (VUS) is where you cannot at the time determine if the variant is disease causing or not.

Phenylketonuria (PKU)

• Phenylketonuria (PKU) is an inborn error of metabolism (MIM #261600).
• Caused by deficiency of phenylalanine hydroxylase activity.
• It can result in impaired cognitive development from a neurotoxic effect of hyperphenylalaninemia if undiagnosed and untreated.
• Newborns are screened for PKU.
• The allelic variant c.1222C>T (protein change R408W; p.Arg408Trp) is a missense variant.
• The mutation leads to amino acid Arg exchange to Trp in position 408, resulting in loss-of-function.
• ClinVar classifies this variant as pathogenic.

Chromosomal Aberrations

• Numerical aberrations relate to aneuploidy include loss or gain of one chromosome (2n-1 or 2n+1).
• Missegregation of a chromosome pair occurs during cell division.
• Changes during the germ cell division are responsible for conditions such as trisomy 21 (Down syndrome).
• Chromosomal aneuploidy are the most common changes seen in humans.
• Rate of numerical aberrations is one per 25-50 cell cycles.
• Numerical aberrations relate to polyploidy includes Euploidy meaning a complete set of chromosomes.
• Chromosome number in haploid is 23 and diploid is 46 chromosomes in humans.
• Polyploidy is 3n (triploidy = 69 chromosomes) or 4n (tetraploidy = 92 chromosomes).
• Failure in meiotic (formation of gametes) or mitotic (after conception) cell division.

Structural Aberrations

• Structural aberrations mean the rearrangement of structure or regional organization of chromosomes.
• Alterations of chromosome structure occur when homologous chromosomes line up improperly, or when chromosome breakage occurs and are not repaired correctly, during cell division.
• Basic types of structural change:
• Deletion: chromosome break and subsequent chromosomes leading to loss, there are two kinds.
• Terminal deletion, where the loss that includes the chromosome's tip is missing.
• An interstitial deletion results when two breaks occur and the material between the breaks is lost.
• Ring chromosome: Deletions sometimes occur at both tips of a chromosomes and the end fuses create a ring.
• Translocation: is where interchange of genetic material between non-homologous chromosomes can happen.
• Balanced translocations represent one of the most common chromosomal aberrations in humans, occurring in 1 of every 500 to 1000 individuals.
• Robertsonian represents a type of basic translocation.
• The resulting chromosomes are called derivative chromosomes.
• Inversion: the result of two breakage on a chromosome followed by the re-insertion of the intervening fragment at its original site but inverted by 180°.
• Pericentric: inversion that includes the centromere.
• Paracentric: inversion does not involve the centromere.
• Duplications: gain of genetic material, which can arise from unequal crossover.

Chromosomal Aberrations in Human Pathology

• Down Syndrome: a particular combination of phenotypic features that includes mental retardation and characteristic facial appearance.
• The cause is Trisomy 21.
• Linked to a Gene dosage of critical region of chromosome 21.
• NFATc is the Nuclear Factor Of Activated T-cells, Cytoplasmic, Calcineurin-Dependent.
• DYRK1A is the Dual Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A.
• DSCR1 is the Regulator Of Calcineurin 1.

Summary

• To use correct, up-to-date terms is important.
• Origin of genetic variation includes spontaneous or induced process of mutation.
• Alteration of DNA sequence is an important aspect when studying genetic conditions.
• Consequences of genetic variation are measured at a molecular and functional level.
• Chromosomal aberrations also play a key role.