Mod 1 & 2

• Types of Congenital Diseases

  Genetic

  →Inherited (eg CF)

  →Spontaneous (eg Trisomt 21)

  Non-Genetic

  →Environmental (eg Rubella malformations)

  →Accidental (eg Cerebal Palsy)
• Types of Acquired Diseases

  Inflammatory

  →Acute (acute appendicitis)

  →Chronic (Tuberculosis)

  Growth Disorder

  →Neoplastic (Cancer)

  →Non-Neoplastic (Prostate hyperplasia)

  Injury without Repair

  →Physical (Bone fracture)

  →Biochemical (Aspirin ulcers)

  Haeomdynamic

  →Shock (Haemorrhagic shock)

  →Blockage (ischaemia)

  Immune Disorder

  →Deficiency (AIDS)

  →Excessive (Allergy, autoimmunity)

  Metabolic & Degenerative

  →Diabetes

  →Osteoarthritis
• Terminological Prefixes


  Cyto = Cell

  Dys = Disordered

  Hyper = in Excess

  Hypo = Deficiency

  Leuko = White

  Meta = Change from one to another

  Neo = New
• Terminological Suffixes


  aemia = of the Blood

  cytosis = Increase in a cell type

  itis = Inflammatory process

  oid = Resembling

  oma = Growth

  opathy = Diseased state

  osis = State or condition

  penia = Lack of

  Plasia = Growth disorder
• Terminology: Types of Pathology

  -Morbid anatomy = post mortem macroscopic examination of disease (since classical greece)

  -Cellular and Histopathology = Microscopic (since 1800 Louis Pasteur) examination of disease

  -Molecular Pathology = Description if disease process in terms of biochemical and molecular biological processes
• Case Study: Cystic Fibrosis

  -Autosomal Recessive mutation CFTR

  →Thick mucous secretions

  →Chest infections

  →Lung damage`
• Case Study: Asbestos


  -Exposure to asbestos fibres

  →Pulmonary and pleural fibrosis

  →Mesothelioma
• Cell Responses Causing Pathological Conditions

  -Degeneration (loss of functionality) or Atrophy (wasteage)

  -Apoptosis or Necrosis

  -Inflammation

  -Regeneration, Hyperplasia, or Hypertrophy

  -Dysplasia (eg fibrosis) or Neoplasia
• Types of Pathologies: Case Study: HER-2 Oncogene

  Her-1 = proto oncogene coding for epidermal growth factor receptor

  →Over expressed in 20-30% of breast cancers (90% of this is in mutated Her 2 (oncogene) form)

  →Herceptin binds to EGFR blocking its action and countering this

  #Only useful where oncogenic Her-2 is at fault

  ←Use FISH to see if cells possess oncogenic Her-2
• Types of Tissue Biospy

  -Incisional = Cut sample of tissue of interest

  -Excisional = Cut out all of tissue of interest

  -Exfoliative Cytology = Smear or brush tissue

  -Core Needle Biopsy = Needle cuts out small chunk of tissue

  -Fine Needle Aspiration = Needle sucks out fluid and cells.
• Examination of Cell Morphology

  -Histology or architecture of tissue and cell types present
• Examination of Cell Microbiology

  -Examine interactions with known microbes and chemical/biological stresses
• Examination of Cell Antigen Expression

  -Immunohistochemistry

  = Staining with labelled antigens to examine a suspension
• Examination of Cell Molecular Analysis

  FISH = Fluorescent in situ Hydrbidisation

  →Using labelled DNA/RNA compliments to locate specific DNA

  →Essentially used in DNA sequencing

  #We also usually mark telomeres as well as gene of interest.
• What is an Adenocarcinoma


  Cancer arising from gland or in which neoplastic cells attempt to form a gland
• Polymerase Chain Reactions


  In vitro DNA replication

  -uses computerised thermal cylinders and thermostable taq polymerase from Thermus aquaticus

  →Denaturation 90-95°C

  →Annealing 50-65°C

  →DNA Synthesis 72°C

  -Substitute for cloning and can be used on small amounts of DNA not usually present in tissue being tested (eg virus)

  -Can be used on highly degraded DNA
• Gel Electrophoresis


  -Allows separation of a population of DNA by size and conformation

  -eg examining offspring against parent, seeing a band moving further = de novo gene deletion
• Types of Gel Electrophoresis Gels

  Polyacrylamide Gels = for ssDNA less than 500nucleotides

  Agarose Gels = More porous for molecules 300-20,000 nucleotides

  Pulse-Field Gel = for V long DNA molecules
• Principles of DNA Sequencing

  Hybridise with mix of normal dTP (deoxyribosenucleoside triphosphates) and different colour fluoro tagged ddTPs

  →the rare incorporation of a ddTP will stop DNA polymerase sliding and stop strand growth

  -Run DNA on gel to separate

  -Read up (biggest migration = shortest)

  #can run on automated using a laser and passing to a detector with sliding gel plate (by using different flouro colours for diff ddTP bases
• Reverse Transcriptase PCR


  -Uses RT to create a cDNA library from the cell of interest's mRNA = Indication of cell's gene expression rather than its full genome

  -PT Steps:

  →make cDNA strand with mRNA

  →Degrade mRNA with RNase H

  →Synthesise compliment to cDNA
• Real Time PCR


  Often used with reverse transcriptase PCR

  →accumulation of products is monitored through cycling to give an indication of initial amount of DNA (/relevant mRNA)

  -TaqMan probe

  →Hybridises with a target amplicon with terminally blocked 3'

  →has 5' Reporter and 3' Quencher, Increasing distance increases Reporter Fluorescence

  -After enough cycles the fluorescence passes a defined threshold at that cycle is called the Threshold Cycle (Ct)
• Real Time PCR: Threshold Cycle

  -Cycle where target amplification is first detect (fluorescence is greater than threshold)

  -The greater the initial quantity of DNA the faster the increase (passes "lag phase") and the lower the Ct

  =Low Ct → High [DNA] initial
• DNA Hybridisation Methods


  -Dot Blot

  -Southern Blot

  -FISH

  -SNP Arrays

  -Micro Arrays

  -Array CGH
• Nouthern vs Southern vs Western Blotting

  Northern = RNA

  Southern = DNA

  Western = Protein

  All involve running on a gel and hybridisation staining
• DNA Hybridisation Methods: Dot Blot


  Functionally similar all the "Compass" Blotting methods, but without electrophoresis

  -Samples are blotted on a membrane and hybridisation stained
• DNA Hybridisation Methods: Southern Blot

  = DNA Fragmentation with Restriction Enzymes

  →Electrophoresis

  →hybridisation staining
• DNA Hybridisation Methods: FISH


  =Fluoro staining with DNA Hybridisation probe into cells

  →Fluorescence microscopy, examining location and presence of DNA on chromosomes and in tissue
• DNA Hybridisation Methods: SNP Arrays


  Types of Micro Array

  -Examination and hybridisation of many genomes/ genes with DNA Hybridisation for specific SNPs of an allele (SNP = Single Nucleotide Polymorphism)

  -Useful for studying slight differences between many similar genes/genomes

  →Use for parallel genetic testing
• DNA Hybridisation Methods: Micro Arrays


  Many genes/ genome segments spotted onto chip

  →Use for parallel genetic testing
• DNA Hybridisation Methods: Array CGH


  "Array Comparative Genomic Hybridization"

  -Comparison of patient genome against a reference and identifies differences
• Rate of Endogenous Mutation

  = Spontaneous errors in DNA replication or repair

  - 1/10 million pase pairs

  →99.9% repaired by proofreading mechanisms

  ~1 nucleotide change per cell division
• Mutation Types


  Monogenic

  Chromosomal

  Complex/Multifactoral
• Chromosomal Disorders


  -Excess or deficiency of genes contained in a chromosome or chromosome segment

  →Translocations, Deletions, Insertions, Duplications, Inversion

  →Chromosome loss or duplication

  -Rate ~ 7 per 1000 liveborn

  -Cause 50% of all spontaneous first trimester miscarriages
• Case Study: Chromosomal Disorders: Patau Syndrome


  -Trisomy 13

  -1 in 12,000 live births

  -Increasing risk with maternal age

  -Rarely survive beyond 1yo

  -Holopronsencephaly (forebrain doesn't divide)

  -Heart Defeces

  -Dysmorphology

  -Seizers

  -Severe mental retardation
• Case Study: Chromosomal Disorders: Turner's Syndrome


  45 XO = Complete or partial chromosome monosomy in a phenotypic female

  →Poorly formed or absent ovaries and incomplete sexual development

  →Infertility

  →Webbed neck and low hairling and ears

  →Broad Chest and widely spaced nipples

  →Nevi (nevus = birthmark)

  →Lymphoedema

  →Short stature
• Case Study: Chromosomal Disorders: Cri du Chat


  Depletion 5p Syndrome

  →Severe mental retardation

  →Monotone, weak, catlike cry

  →Heart Defect

  →Microcephally

  →high palate, round face with small receding chin

  →Wide spaced eyes

  →low ears and wide low nose

  →Distinct Palmar Creases

  →Epicanthic eyelid folds
• Monogenic Mutations


  -Caused by single mutant genes

  -Rec or Dom

  -Mito or Nuclear

  -Obvious pedigree patterns usually

  -~2% of population

  -Subsitition, Deletion, insetion
• Monogenic Mutations: Substitution


  -Transition: Pur↔Pur, Pyr↔Pyr

  -Transversion: Pur↔Pyr

  -Due to larger number of possibilities we -would expect transversions to be more common but there is a C→T hotspot (UV radiation)

  →Synonymous/silent = no AA change due to wobble

  →Nonsense = Becomes Termination Sequence

  →Missence = Becomes different AA

  (conservative or non conservative depending on AA properties)
• Monogenic Mutations: Autosomal Dominant


  ~50% of Mendelian diseases Also anc child has a 50% risk

  -1 in 500 familial hypercholsterolaemia in euro or japanese

  -1 in 100 myotonic dystrophy in N america

  -1 in 2500-300 of huntington disease, neurofibromatosis, and polycystic kidney disease
• Monogenic Mutations: X Linkage

  Incidence higher in males

  -Heterozygotes are usually unaffected or to a lesser extend due to random X inactivation (milder disease)

  eg

  →Androgen Insensitivity Syndrome

  →Muscular Dystrophy

  →Fragile X Syndrome

  →Haemophilia