Genetics Overview: Mutations and Abnormalities

Dilute Colour Mutation

• An autosomal recessive condition
• dd results in a diluted colour
• D- results in normal colour
• CRICOS code 00025B

Dosage Compensation and Tortoiseshell Cats

• CRICOS code 00025B

Mutations and Abnormalities

• CRICOS code 00025B

The Genetic Translation Code

• Three nucleotides code for one amino acid (codon)
• Start and stop codons signal the beginning and end of the amino acid chain
• DNA genetic code uses adenine, thymine, cytosine, and guanine (ATCG)
• In mRNA, thymine (T) is replaced with uracil (U)
• CRICOS code 00025B

Mutations

• Changes in DNA or RNA
• Single base substitution in coding regions can be silent (synonymous), missense (non-synonymous), or nonsense (stop)
• Indel: insertion or deletion of one or a few nucleotides, can lead to frameshifts within coding regions
• A splice site mutation alters nucleotides at splice control sequences changing the patterns of RNA splicing
• CRICOS code 00025B

Muscular Dystrophy in Golden Retrievers

• A splice site mutation in intron 6 causes a complete deletion of exon 7 from mRNA
• Leads to a frameshift in following exons
• Loss of dystrophin from muscle
• CRICOS code 00025B

Defective Chromosomal Recombination/Segregation

• Abnormalities can arise when chromosomes rearrange or fail to segregate properly
• Numerical anomalies result in aneuploidy, either the addition or loss of individual chromosomes
• Structural abnormalities are rearrangements of genetic material within or between chromosomes
• Can be balanced or unbalanced
• CRICOS code 00025B

Sex Chromosome Aneuploidy

• Female offspring can have the following:
  • XX
  • X0
  • XXX
• CRIGOS code 00025B

Loss or Gain of a Single Chromosome

• Normal = euploidy = having the normal number of chromosomes
• Abnormal = aneuploidy = having fewer or more than the normal diploid
• Most common forms of aneuploidy are monosomy (loss of one of a pair of chromosomes) and trisomy (three chromosomes instead of a pair)
• Effects of Aneuploidy of autosomes: Serious developmental problems, typically lethal = embryo/foetal loss i.e not passed on. Trisomies of small chromosomes can lead to live animals
• Effects of polyploidy in mammals: In mammals, polyploidy is lethal, usually at embryonic/foetal stage, causes severe developmental abnormalities
• CRICOS code 00025B

Abnormal Chromosome Structure

• Changes to the structure of chromosomes caused by mistakes in meiotic recombination and DNA break repairs
• Balanced changes: do not change the overall DNA content of a cell, although the gene neighborhood is affected and might alter gene expression.
• Unbalanced changes: overall loss or gain of total amount of DNA
• CRICOS code 00025B

Reduced Fertility due to Translocations

• Complex arrangements of chromosomes during meiosis
• Translocations can be inherited through balanced gametes
• Translocations can lead to creation of balanced but unbalanced zygotes
• Translocations can lead to reduced fertility
• CRICOS code 00025B

Reciprocal Translocation – Cause of Low Fertility

• Individual has reciprocal translocation during meiosis
• Balanced individual = has full complement of all chromosomal pieces
• All gametes are functional, but some are unbalanced
• Upon fertilization with normal gamete, some zygotes are unbalanced and die
• Reduction in fecundity
• Changes are inherited through two gametes with balanced translocation
• CRICOS code 00025B

Robertsonian Translocation

• Same concept as Reciprocal Translocation
• CRICOS code 00025B

Abnormal Chromosome Structure

• Genome rearrangement (inversion and translocation). Occurs during meiosis. Can lead to new functions or to lethal loss of function.
• CRICOS code 00025B

Mutation and Repair

• CRICOS code 00025B

Mutation Heritability Depends on Mutation Location

• Somatic mutations:occur in non-reproductive cells passed onto daughter cells. Creates a clone of mutant cells - More widespread if occurring in embryo
• Germ-line mutations: occur in cells that produce gametes passed to future generations. All cells in the offspring’s body carry the mutation
• CRICOS code 00025B

Genetic Tests

• CRICOS code 00025B

Sample Collection, DNA/RNA Extraction and Amplification

• Avoid contamination and store/ship appropriately
• DNA is stable, RNA is not
• PCR (Polymerase Chain Reaction) is a technique used to amplify DNA segments
• Real-time PCR can be used to quantify gene expression or DNA copy number
• CRICOS code 00025B

PCR Process

• Three steps:

  • Denaturation: heating DNA to separate the strands
  • Annealing: primers bind to specific DNA sequences
  • Extension: Taq DNA polymerase adds dNTPs to create new complementary strands

• Repeated cycles amplify the target DNA

• CRICOS code 00025B

Agarose Gel Electrophoresis

• Used to separate DNA fragments based on size
• Fluorescent dye that binds DNA eg ethidium bromide or SYBR Safe is added to the gel
• DNA fragments are visualized under UV light
• CRICOS code 00025B

Real-Time Quantitative PCR (qPCR)

• Measures DNA amplification in real-time
• CT value = threshold cycle, a measure of the starting amount of DNA
• Used for presence/absence (Syber or probes) or quantitative
• CRICOS code 00025B

DNA Sequencing

• “Gold standard” for detecting single or multiple nucleotide substitutions
• Provides detailed information about nucleotide changes
• CRICOS code 00025B

Gene Types in Multifactorial Disease

• Disease susceptibility genes: gene sequence variants associated with an increased risk of disease but may not cause the disease on their own
• Modifier genes: genes whose expression or function can influence the phenotype resulting from mutation of another independent gene
• Mutations: Heritable detrimental sequence changes in a gene that are commonly associated with disease
• Polymorphisms: Sequence variation (point mutation) among individuals that are common in a population (>1%) and that are usually not associated with disease
• CRICOS code 00025B

Testing for Multifactorial Diseases/Traits

• If all the contributing genes, their interactions and environmental effects are known … then genetic testing could be undertaken as we do for Mendelian traits. But, we usually don’t know all, or even all the major, contributing genes, causative or associated gene variants
• Therefore we measure risk of associated variants … Can use Odds Ratios
• OR>1 indicates that the condition or event is more likely in the first group than the general population
• OR<1 indicates that the condition or event is less likely in the first group than the general population
• The closer the OR is to 1, the smaller the difference in effect between the two groups
• CRICOS code 00025B

Quantitative Trait Loci (QTLs)

• Same principles from multi-factorial disease
• Use the term QTL when identified chromosome region but not actual ‘causative’ mutation
• A QTL can be represented by the actual mutation if it is known (direct test) or SNP/microsatellite marker (indirect test)
• Each QTL marker tests only for a proportion of the genetic contribution to that trait.
• CRICOS code 00025B

Cancer Genetics

• CRICOS code 00025B

Cancer is a Multi-Gene Disorder

• Cancer arises from the accumulation of mutations in multiple genes
• A single mutation in a single gene is not sufficient to cause cancer
• Familial cancer: germ-line mutation
• Sporadic cancer: somatic mutation
• CRICOS code 00025B

Cancer Cell Characteristics

• Defective response to DNA damage
• Defective telomeres
• Uncontrolled cell proliferation
• Immortalization
• Defective apoptosis
• Induction of angiogenesis
• Increased cell motility
• Reduced cell adhesion

Mitochondrial Disease

• CRICOS code 00025B

Mitochondrial DNA

• Replicates separately from nuclear DNA
• Codes for metabolic proteins (ATP synthesis, fatty acid metabolism, citric acid cycle)
• Many essential genes for mitochondrial replication and function have been transferred to the nucleus
• Nuclear genes are “protected” from mutagenic environment of mitochondria
• CRICOS code 00025B

Human mtDNA Map

• 12S & 16S rRNAs
• Cytochrome b
• D-loop
• tRNAs
• Overlap of ATPase 6 and 8
• CRICOS code 00025B

Maternal Inheritance of Mitochondrial DNA

• Uniparental (maternal) inheritance of mitochondria and hence mtDNA
• Both sons and daughters inherit the mtDNA of their mother
• Mitochondrial inheritance is thus “non-Mendelian” (ie not derived from both parents)
• CRICOS code 00025B

Nuclear-Encoded Mitochondrial Genes

• Many essential genes for mitochondrial replication and function have been transferred to the nucleus
• Nuclear genes are “protected” from mutagenic environment of mitochondria
• Nuclear-encoded mitochondrial proteins synthesized in cytoplasm and imported into mitochondria facilitated by a signal peptide (SP)

High Mutation Rate in mtDNA

• Reactive oxygen species (ROS or free radicals) produced during oxidative phosphorylation
• Low efficiency proof reading capacity of the gamma DNA polymerase
• CRICOS code 00025B

Mitochondrial Disease

• Disease pathogenesis relates to oxidative phosphorylation … the process that produces energy
• Mitochondrial diseases show delayed onset and progressive decline
• CRICOS code 00025B