Genetics Flashcards
What cells are used in cytogenetic analysis?
Any nucleated cells, blood most common
How do you do cytogenetic analysis?
Centrifuge blood Hypotonic solution to lose rbc Stimulate wbc division with mitogen PHA-phylohaemiagglutinin Metaphase block called colchicine Drop from height to see banding patterns Stain Microscope
Types of germ line defects?
Chromosomal defects
Mitochondrial disorders
Monogenic disorders
Polygenic disorders
Types of somatic defects?
Cancer
Mosaics
Chimeras
How do you get banding on chromosomes?
Treat with protease like trypsin which attacks chromatin so when stained bands show
What does banding on chromosomes show?
The density of genes- light bands means more genes
What does a missing band mean?
Chromosomal defect
What are acrocentric chromosomes?
Don’t have a short arm
Repeat units only
What does FISH stand for?
Fluorescence in situ hybridisation
Why can we use FISH?
We know the sequence of the human genome
What is FISH used for?
Detecting more subtle defects..chromosomal abnormalities
How is FISH carried out?
Localises a region of DNA in a chromosome
DNA probe labelled with fluorescent dye
Denature probe and hybridise with patients sample
Fluorescent microscope
What are centromeres probes?
Repetitive sequences found around the centromere of a specific chromosome
Diagnosis of trisomies using sample of chorionic villi
What are chromosome specific unique sequence probes?
Specific for a single locus
Submicroscopic deletions and duplications
What are telomeric probes?
Simultaneous analysis of the subtelomeric region of every chromosome
Identifies tiny cryptic subtelomeric abnormalities
What are whole chromosome ‘paint’ probes?
Cocktail of probes obtained from different parts of a chromosome
Relevant chromosome fluoresces
Subtle translocations and identifying the origin of additional material
Name the three types of autosomal abnormalities
Numerical
Structural
Sex chromosome
What is polyploidy?
Extra whole complement of chromosome…triploid
Lethal
What is monosomy?
Missing one chromosome
Not compatible with normal growth
What is trisomy?
Extra copy of one chromosome
Which chromosome has an extra copy in Down’s Syndrome?
Chromosome 21
Why can people with Down’s survive?
Smaller chromosome so less genes to mess up genome
How is Down’s caused?
Non-dysjunction in female meiosis
What is a Robertsonian translocation?
Two acrocentric chromosomes lose their short arms and fuse together
What is a Reciprical Translocation?
Material exchange between homologous chromosomes
What happens if someone inherits balanced translocations?
Often no effect
Can produce offspring with unbalanced chromosomal complement
Can give partial trisomy or monosomy
What will a visible deletion most likely cause?
Mental retardation
Klinefelter’s syndrome
XXY males
Turner’s Syndrome
XO females Sterile Short stature Webbed neck Only monosomy compatible with development
How many copies of DNA in each mitochondria?
2-10 copies circular DNA
Mitochondrial DNA maternally or paternally inherited?
Maternally
What does heteroplasmic mean?
Mutation in some mitochondrial DNA
Affects how strong phenotype is
Can pass to some offspring but not others
Severity of disease can change over time
What is LHON?
Leber's hereditary optic neuropathy Mitochondrial mutation Mutation in NADH dehydrogenase Optic nerve damage Blindness
What is Leigh’s syndrome?
Mitochondrial mutation
Mutation in ATP Synthase
Degeneration of basal ganglia
Problem with nervous system
What is Pearson’s syndrome?
Mitochondrial mutation
Deletion of mitochondrial DNA
Bone marrow failure
Problem with immune system and blood
What is a monogenic disorder?
Defects in a single gene
Simple, Mendelian patterns of inheritance
What is penetrance?
Chance of inheriting disease if carrying the mutation
Genetic background determines if you have mutation
Some diseases can be decreased with changing diet and environment
What is expressivity?
Variation in phenotype in affected individuals due to genetic background
What is genomic imprinting
Expression of alleles depends on which parent they are inherited from
Only affects minority of genes
Name some imprinting disorders
Prader-Willi syndrome
Angelman syndrome
Beckwith-Wiedemann syndrome
What is the hardy-Weinberg principle?
Allele and genotype frequencies remain the same unless specific disturbing influences are introduced
What are the disturbing influences regarding the hardy-Weinberg principle?
Non-random mating Mutations Selection Random genetic drift Gene flow
What is the hardy-Weinberg equation?
P+q=1
p2+q2+2pq=1
Homozygous AA=p2 aa=q2
Heterozygous Aa=2pq
What is positional cloning?
Disease Map position of gene Gene Gene product Functional studies From genotype to phenotype
What is linkage analysis?
Based on genetic distance
Find genetic marker coinherited with disease
Markers 10^6 apart have 1% recombination frequency meaning they are likely to be coinherited
Polymorphic markers vary person-person and chromosome-chromosome
Measure linkage by LOD score…>3 indicates linkage
What are RFLPs?
Restriction fragment length polymorphisms
Polymorphic marker
Single base changes - gain/loss or restriction site
What are VNTRs?
Variable number tandem repeats
Polymorphic markers
20-30bp repeats
What are microsatellites?
Used in fingerprinting
Di, tri, tetra nucleotide repeats
What are SNPs?
Single base changes occur every 300bp
Linkage analysis
Pre-genome project how was gene mapping done?
Regional genetic map
Markers to select genetic clones
Produce a contig
What is a contig?
Overlapping fragments of cloned DNA representing the entire region
What is a contig used for?
Screen cDNA libraries Search for CPG Islands Zoo blotting Exon trapping CDNA selection
How is gene mapping done now?
Genetic maps available Thousands mapped genes and Expressed Sequenced Tags Genes predicted from genomic sequence Rapid identification of candidate genes Computers used
Symptoms of cystic fibrosis
Chronic lung disease Bowel obstruction Pancreatic failure Increased sweat electrolytes Premature death
How was the CF gene isolated?
RFLP linkage on 7q31
MET is a target kinase receptor, it is close to CF gene but need even closer marker
Chromosome jumping and walking
Properties of CF gene
250kb
27 exons
6.5kb mRNA
1480 amino acids
In CF, what protein is affected?
Cystic fibrosis transmembrane conductance regulator
Transmembrane protein
ATP binding site
Chloride channel when fails increases salt conc making thicker mucus
How is the CF protein mutated?
Mainly phenylalanine deletion
Functional analysis of CF
Cell culture studies to create transport defects
Knockout mice to develop correct pathology to disease when exposed
What is Duchenne Muscular Dystrophy?
X-linked recessive
1:4000 boys
Death before 30
chromosome translocations through Xp21 in girls, deletion in boys
Molecular studies of DMD
Xp21 probes - one loosely linked marker
Cloned Xp21 translocation - tightly linked marker
Subtractive cloning using Xp21 deletions to mark out what was missing - close marker
DMD gene
2000kb >65 exons 16kb mRNA 368 amino acids Mutations common
Protein involved in DMD
Dystrophin
427kb
Features in common with muscle structural proteins
0.002% muscle protein
Links actin to protein complex in plasma membrane
Links muscle cells to basal lamina
Mutations in DMD
Large deletions (highly truncated protein) causes severe disease Small deletions so only partly truncated causes mild disease
Functional analysis of DMD
Mice have dystrophin deletion didn’t show symptoms
Eventually diaphragm disintegrated as that muscle worked the most
Failure of repair so degradation
Features of Huntington’s disease
Autosomal dominant
Progressive neurodegeneration
Late onset
First disease to be mapped by linkage 1983
Mutation in Huntington’s
Normal 16-36 repeats
HD 42-86 repeats
First example of trinucleotide repeat expansion
What are trinucleotide repeat disorders?
All involve (XYZ)n sequence
N is increased
All have same markers
Severity increases through succeeding generations
Can be mitosis/meiotically unstable - increase expansions
How does repeat expansion cause disease in fragile sites?
(CGG) >200
Increase methylation so decrease transcription
How does repeat expansion cause disease in neurodegenerative disorders?
(CAG) <150
Multiple effects on protein-protein interactions and toxic protein aggregates
How does repeat expansion cause disease in myotonic dystrophy?
(CTG) 200-400
In 3’ untranslated region - direct RNA mediated effects
Neuromuscular system affected
How does repeat expansion cause disease in Friedrich’s ataxia?
(GAA) 200-900
In intron - decreased transcription of Frataxin
Affects mitochondrial protein
Structure of Sickle Cell Anaemia
Autosomal recessive Single base change Heterozygous advantage of malaria resistance GAG to GTG beta globin defect Haemoglobin precipitates
What are some other abnormalities in the structure of haemoglobinopathies?
Point mutations Deletions - uneven crossover in meiosis Insertions making stronger globin chains Frame shifts Chain termination to make longer chains Fusion chain where two Hb stick together
What are thalassaemias?
Single gene disorders
Autosomal recessive
Decreased synthesis of one or more Hb chains
If all 4 alpha Hb genes deleted in alpha thalassaemias?
Alpha0 homozygote - gamma 4 - death
If 3 alpha Hb genes deleted in alpha thalassaemias?
Alpha0/alpha+ compound heterozygote - variable anaemia
If 2 or 1 alpha Hb genes deleted in alpha thalassaemias?
Alpha0 heterozygote
Alpha0+ homozygote - mild anaemia
Alpha0+ heterozygote
How are beta thalassaemias formed?
Beta0 complete lack of B chains
Beta+ decreased B synthesis
Not due to deletions
Nonsense mutations- premature termination
Promoter mutations- decrease transcription
Defective splicing- decrease translation
What are gamma/beta thalassaemias?
Decreased synthesis of gamma and beta chains
Rare
Variable anaemia
Mild form is hereditary persistence of foetal Hb
What are polygenic disorders?
Many genes
Unclear inheritance
Many factors
What does epistasis mean?
Interaction between genes acting in a common pathway
What is genetic heterogeneity?
Alternate genes in different pathways
What is the liability model?
All contributing factors in disease termed liability
Population shows normal distribution
Above threshold disease develops
Genetic factors shift curve right increasing chance of disease
What is heritability?
Proportion of risk attributable to genetic factors
Schizophrenia 81%
Type2 diabetes 26%
Problems with genetic analysis
Not all susceptible individuals affected due to environmental factors
Genetic heterogeneity- don’t know what genes actually affected as many susceptible
Phenocopy - disease purely from environment
Parametric vs non-parametric linkage analysis
Parametric - difficult to apply as assume pattern of inheritance and don’t know if dominant or recessive
Non-parametric - more successful, do sufferers share common part of DNA that may carry disease gene
What are association studies?
Genome wide association studies
- SNPs
- HapMap to see if inheriting in blocks
- thousand genome project
Schizophrenia using linkage analysis
Problems of phenocopy and low penetrance in families
Two linkages now refuted
Suggest linkage to 6p
Schizophrenia using GWAS
Copy number variations at 1q, 15q, 22q
6p-HLA region so perhaps some autoimmune disease
1000s common variants have small effects, difficult to use data
How are genes identified in type 1 diabetes using non-parametric allele sharing?
See if individuals inherit same chromosomal region
Whole genome scan to pick out common regions associated with disease
How are genes identified in type 1 diabetes using animal models?
Inherited diabetes in mice
Look for homologous genes in man as genomes very similar
How are genes identified in type 1 diabetes using GWAS?
> 40 loci identified
6p21 HLA region most important
Genetic changes more likely to cause disease
Different genes affected in individuals to give a type of diabetes
How is the gene identified in type 2 diabetes?
Linkage analysis unsuccessful as genetically complex
Rare inherited forms
Many GWAS done as big problem
HLA not important - not auto immune disease
No main predisposing loci
Alleles discovered to increase risk by small a,punt especially if all together but mainly diet
What is familial hypercholesterolaemia?
Autosomal dominant Monogene disorder Mutation in LDL receptor gene Heart attacks in children 30 loci associated with circulating lipid levels....lipid metabolism important in cardio disease
Genes associated with hypertension?
Angiotensin gene causes blood vessel constriction
Polymorphism in angiotensin converting enzyme linked to risk of heart attack
GWAS 14 loci identified
What is Gestmann-straussler disease?
Autosomal dominant
PrPc mutation
Could get somatic mutations in gene like cancer
What is Alzheimer’s disease?
Senile dementia
Genetic predisposition
Neurofibrillary tangles and amyloid protein plaques which are pathogenic
Age related
Genes involved in Alzheimer’s disease?
Plaques from amyloid precursor protein - APP on chr 21 - mutations in early onset families
Presenilin 1 on chr14 and presenilin 2 on chr1 - mutations in early onset families
Apolipoprotein E on chr19 - lipid transport, late and early onset, 3 major alleles with E4 giving increased risk
Epistasis and genetic heterogeneity evident
Most late onset no genetic origin
What is the mechanism for Alzheimer’s disease?
APP into fragments by B secretase
Fragments form AB peptide by presenilin
Peptide forms plaques with apoliopoproteins
Neuronal death
What is prenatal testing used for?
Monogenic defects and Down’s syndrome
What is amniocentesis?
Remove amniotic fluid and grow foetal cells
16-18 weeks gestation
Takes weeks to grow cells
Increased miscarriage
What is chorionic villus sample?
Remove part of chorionic villus
11-12 weeks gestation
Immediate analysis
Sample divides rapidly
What is neonatal testing used for?
Diagnosis and blood screening
Heel prick
Tests for phenylketonuria as can’t break down phenylalanine
What is adult testing used for?
Carrier detection
Pre-symptomatic diagnosis of late onset disease
Eg. Huntington’s
What is molecular analysis?
PCR based investigations of specific genes
Indirect determination
DNA sequencing to look for deletions or truncated PCR products
Point mutations if there is a known mutation
Unknown mutations using screening methods like SSCP and sequencing
How are point mutations found using molecular analysis?
Amplification refractory mutation system - PCR primers specific for mutations
Loss/gain of restriction enzyme site - can pick up polymorphisms
Allele specific oligonucleotides can be hybridised to PCR products
Sequencing
When is the indirect determination method used in molecular analysis?
Gene not cloned
Relies on genetic linkage
Allele linkage due to founder effect, polymorphism always associated with mutation
Locus linkage- work out linkage of mutant allele to a polymorphic marker in a family, can be unreliable
Conventional treatment for genetic disease?
Physiotherapy and antibiotics for cystic fibrosis
Expands lifespan but doesn’t cure
Environmental modification to treat genetic disease?
Avoidance of sunlight for xeroderma pigmentosa
Surgery to treat genetic disease?
Correction of virilisation in girls with congenital adrenal hyperplasia
Plastic surgery for Down’s- controversial
Metabolic manipulation to treat genetic disease?
Restricting phenylalanine intake in patients with phenylketonuria
Prevents retardation from build up of metabolites
Gene product replacement to treat genetic disease?
Insulin for diabetes
Factor VIII administration for haemophiliacs
Tissue and organ transplant to treat genetic disease?
Natural - heart and lung in CF
- bone marrow for thalassaemias to help symptoms
Artificial - neoorgans experimental
Stem cell therapy to treat genetic disease?
Pluripotent stem cells - embryonic (ethical)
- induced stem cells by putting differentiated cell in mixture of 4 genes
Multi potent stem cells- many cell types but not all
- mesenchymal stem cells
What is gene therapy?
Correct gene defect with with transgene
Replace gene with homologous recombination- theoretical
What are viral vectors in gene therapy?
Retroviruses, lentiviruses
Role to take over cell machinery so efficient
Safety problems - must be replication deficient and have insertional mutagenesis
What are the physical methods of gene therapy?
Liposomes
Direct DNA injection
Receptor mediated endocytosis
Can’t cross infect so safe
What are the problems of gene therapy?
Producing and sustaining high level transgene expression
Foreign gene may be inactivated
