GiM Lecture 1,2,3 Flashcards
Reasons for clinical genetics referrals
Child
Birth anomalies, dysmorphic features, learning difficulties
Adult
Diagnosis, predictive testing, carrier testing, family history, foetal loss/recurrent miscarriage
Pregnancy
genetic disorder, screening abnormality
What is genetic counselling
Education process to assist affected or at risk individuals understand the nature of genetic disorder, transmissions and available options
How are genetic diagnosis made
Family tree - pattern of inheritance
Physical examination
Genetic Tests - Chromosome, Genes
Testing For genetic disorders
- Non genetic tests
- Genetic tests
Blood tests
- Enzyme assays
- Inborn errors of metabolism
Haematology
-thalassaemia
X Rays
-Skeletal dysplasia
Genetic Genomic architecture -Cytogenetics -Array based techniques Gene faults -Sequencing -OLA assays -MLPA tests
Advantages and disadvantages of genetic testings for disorders
Advantages
- Early diagnosis
- Early interventions
- Carrier testing
- Reproductive choices
- Prenatal testing
Disadvantages
- no treatment
- does knowing help
What is Pharmacogenomics
Analysis of entire genomes of a group
aim to identify genetic factors influencing response to a drug
What is pharmacogenetics
Study an individuals genetic make up to predict drug response and guide prescription
What is Glevec
Treatment for CML (chronic myeloid leukemia)
Targeted at novel fusion protein which is the product of translocation in CML
What are contributions to human diseases
genetic
environmental
Define
- Multifactorial genetic disorder
the interaction of multiple genes (genetic predisposition) in combination with environmental factors eg type II diabetes, ischemic heart disease.
define
- single gene genetic disorder
a mutation in a single gene = Mendelian inheritance – AD, AR, XL eg cystic fibrosis
Define -
Chromosomal genetic disorder
an imbalance or rearrangement in chromosome structure eg aneuploidy, deletion, translocation
Define
- Mitochondrial genetic disorder
a mutation in mitochondrial DNA
Define
- somatic mutations
mutation(s) within a gene(s) in a defined population of cells that results in disease eg breast cancer
What are the single gene modes of inheritance
Autosomal Dominant
Autosomal Recessive
X-linked
Mitochondrial
What is Autosomal dominant inheritance
- What does it mainly affect
- give 3 examples of autosomal dominant conditions
A trait or disease runs from one generation to the next
Males and females equally affected
Offspring of affected person has a 50% (1 in 2) chance of inheriting the mutation
Structural proteins, receptors, transcription factors
Myotonic dystropy
Marfan Syndrome
Huntingdon Disease
Autosomal Dominant Inheritance
- What genotype are those affected
- What is the change off spring is
a) affected
b) unaffected
Affected = 50% Affected = heterozygous
Unaffected - 50%
Homozygous
What is Penetrance?
- give example of a disease with 100% penetrance
The frequency with which a specific genotype is expressed by those individuals that possess it,
e.g huntingdons has 100% penetrance
What is expressivity
variation in expression - the extent to which a heritable trait is manifested by an individual
e.g. marfan - aortic dilation –> stretch marks
What is anticipation?
- two disease examples
the symptoms of a genetic disorder become apparent at an earlier age as it is passed from one generation to the next. In most cases there is an increase in the severity of symptoms too
- myotonic dystrophy
- huntington’s disease
Define
De Novo Mutation
new mutation that has occurred during gametogenesis or in early embryonic development.
Explain Autosomal Recessive inheritance
- give examples of autosomal recessive inheritance diseases
- Disease seen in one generation
- consanguineous relationship increases risk of disease offspring
- relatives maybe carriers
- affects M and F equally
- Gene mutations not chromosomes
- cystic fibrosis
- metabolic disorders
- haemachromatosis
- sickle cell
Autosomal Recessive inheritance
- What genotype are affected
- What risk of being affected
- risk of being an unaffected carrier
Affected = homozygous or compound heterozgous
Affected = 25%
unaffected Carrier = 2/3
unaffected = 25%
X linked inheritance
- Explain
- Example diseases
Males affected
Females - spectrum - unaffected –> fully
M usually more affected
Female transmission ONLY
Possible in
- Gene mutations, chromosome deletions and duplications
Duchenne Muscular Dystrophy
Fragile X syndrome
Red/green colour blindness
Haemophilia
X linked inheritance - Carrier Mother
- Affected Boy
- Carrier Girl
- Normal Boy
- Normal Girl
25%
X linked inheritance - affected father
- daughters?
- Sons?
D = All carriers S = unaffected - no male transmission
X linked inheritance
Why can females have varying phenotypes
X inactivation
XL dominant vs XL recessive inheritance
CANNOT predict female phenotype in prenatal testing
What is Lyonisation?
- X- inactivation
- One X chromosome in cells with XX is turned off.
- Occurs in early embryogenesis
- Most but not all inactivated X genes turned off.
X inactivation and disease expression
What is skewed X inactivation?
What is tissue variability
Skewed = random preference for normal X chromosome to be inactivated over abnormal X
Tissue - Random prefernece for mutated X chromosome to be active in crucial tissue group
X linked Dominant inheritance
- Affects?
- Who does Rett syndrome effect
- Who does Fragile X syndrome effect
Males and females
Rett - lethal in M, phenotype only in F
Fragile X - M affected, F - spectrum
X linked Recessive
- affects?
- are carrier girls affected?
Affects - males,
carrier females
can be affected F is homozygous
carrier girls - affected if x inactivation skews
Mitochondrial Inheritance
- explain
- example
- Maternally inherited diabetes and deafness
- Rare , M = F affected
- only 27 genes in mitchondrial DNA
- always maternally inherited
- affected mother = ALL offspring affected
- affected father - no affect
Family trees What does .... mean? - Circle - square - diamond - diamond crossed out - triangle - triangle crossed out - filled circle/square - square/circle with dot
- female unaffected
- male unaffected
- unknown sex
- still born unknown sex
- miscarriage
- abortion
- affected F/M
- asymptomatic carrier
What is the symbol for
- Consanguinity
How does it affect genetics
Double line between couple
- blood relative
- congenital birth defect 5-6% risk (pop normal = 2-3%)
What is Polymerase chain reaction
In Vitro synthesis of large amounts of DNA
Oligonucleotides define boundaries of synthesis
DNA synthesized by DNA polymerase enzyme
1) Heat denaturation 94 degree
2) primer annealing 55 degree
3) primer extension 72 degree
Products separated by GEL electrophoresis and visualized with UV
What can be determined by PCR
Presence/absence of something
- allele specific PCR
product size by gel electrophoresis
- Oligonucleotide ligation assay
- direct sequencing
How does gel electrophoresis work
Electrical current
small products move towards +ve end
large products remain at -ve end
DNA is -ve charged therefore moves towards +ve
What is the mutation for the most common inherited cause of deafness
Connexin 26
GJB2
What can a delta35g assay show
accurate size analysis - can show single base deletions
- able to distinguish
hetero zygotes, homo zygotes, and normal
What is Allele specific mutation
detection
- method
- use
- distinguish between 2 alleles that differ by one nucleotide
aka - Oligonucleotide Ligation Assay - allele specific primer- 3’ nucleotide base pair with changed nucleotide
- reaction will not occur if base pair not perfect
- able to tell abnormal from normal alleles
How can Cystic fibrosis gene mutations be identified
Cystic Fibrosis genotyping assay
What is Multiplex Ligation- dependent probe amplication analysis (MLPA)
- detects chromosomal DNA copy number changes in multiple targets
What is the amount of product from PCR proportional to?
the amount of target DNA present
What are the problems with mutation anaylsis
gene might be too big for PCR
gene may be repetitive
GC rich regions are hard to PCR
Souther Blotting is used for what?
Fragile X syndrome
DNA
what is DNA sequencing
Detection of unknown mutations
= exact position of mutation
- type of mutation
uses specific PCR product as template
What nucleotides are used in sequencing?
dNTP deoxyribose nucleotides
dideoxyNTP dideoxyribosenucleotides
how do ddTTPs help with sequencing
stop chains continuing to grow
lots of chains different lengths
can read sequence by gel electrophoresis
What is MEGF10 mutation screening
distinguish between different muscular dystrophies
What is clonal sequencing
next generation
Illumina Method
Long PCR product covering gene
produces flow cell images
