Introduction, basics & techniques Flashcards
Reasons for genetic referral
For diagnosis/investigation Family history Fetal loss Recurrent miscarriage Strong family history of cancer
Broad types of testing
Prenatal
Carrier
Predictive
Diagnostic
How to make a genetic diagnosis
Family tree
Physical examination
Genetic tests: chromosomes (karyotype), genes (DNA testing)
Diagnostic genetic test categories
Non-genetic or genetic
Examples of non-genetic tests
Bloods - enzyme assay, haematology
X-rays - skeletal dysplasia (achondroplasia)
Examples of genetic tests
Genomic architecture - cytogenetics, array based techniques
Gene faults - sequencing, OLA assays, MLPA tests
Pharmacogenomics definition
Analysing entire genomes, across groups of individuals, to identify the genetic factors influencing responses to a drug
Pharmacogenetics
Studying an individual’s genetic make up in order to predict responses to a drug and guide prescription
Drug treatments targeted at genetic faults
Somatic e.g. Gleevec
Germline e.g. RP65
Achondroplasia
Affects height - single gene but different heights
Single gene modes of inheritance
AD
AR
XL
Mito
AD Inheritance affects:
Structural proteins, receptors, transcription factors
AD Inheritance applies to:
Myotonic dystrophy
Marfan syndrome
Huntington Syndrome
Chromosome deletion&duplication e.g. 22q11 deletion syndrome
Penetrance definition
The frequency with which a specific genotype is expressed by those individuals that posses it, usually given as percentage e.g. 100% Huntington by 80 y.o. but 80% with BRCA1
Expressivity definition
Variation in expression - the extent to which a heritable trait is manifested by an individual e.g. Marfans differences or BRCA1 good cause ovarian or breast
Anticipation definition
The symptoms of a genetic condition become apparent at an earlier age from one generation to the next - myotonic dystrophy, Huntington’s
New dominant / de novo
A new mutation that has occurred during gametogenesis or early embryonic
AR diseases e.g.s
CF
Many metabolic
Haemachromatosis
Sickle cell disease
XL
Males affected; maybe female from mild to fully DMD Fragile X Red/green colour-blindness Haemophilia No male-male transmission
Influences to XL expression of phenotype
X inactivation
XL dominant vs XL recessive
Lyonisation
Random X-inactivation in cells with more than one X chromosome which compensates for the presence of the double X gene dose - makes a Barr body
X-inactivation
Common if two or more X chromosomes
Early in embryogenesis
Random X silenced
Remains inactive forever
Types of X-inactivation
50% of cells express the normal gene
Skewed X-inactivation
Tissue variability
Skewed X-inactivation
Random preference for normal X chromosome to be inactivated - significant phenotype
Tissue variability
Random preference for the X chromosome with the mutation to be active in crucial tissue group e.g. DMD
XL Dominant (rare)
Rett Syndrome - not compatible with life in males, phenotype only in females
Fragile X syndrome - females range from asymptomatic to fully symptomatic (due to X-inactivation)
XL recessive
Red-green colour blindness
Haemophilia
DMD
Carrier girls usually unaffected but can be significantly because of X-inactivation
Mitochondrial inheritance
Maternally inherited diabetes Deafness Rare Equal in M+F Passed on through mothers egg Highly variable expressivity All children of man unaffected
Asymptomatic carrier sign
Filled in circle inside
Double line
Consanguineous relationship
Splits in two from one origin on tree
Twins
Identical twins have a connecting line making it a triangle
Diamond in family tree
Unknown sex
Consanguinity
Couples who are blood relatives
Share recessive gene mutations
Risk of congenital birth defect (5-6%) vs (2-3%)
Seen in all ethnic groups
Genetic testing for a mutation we already know of
Simple and cheap analytical methods can be used
Genetic testing for a mutation we do not know identity of
Discovery methods (e.g. Sanger DNA sequencing) Clonal sequencing
Polymerase chain reaction (PCR)
In vitro synthesis of large amounts of DNA by copying small starting quantaties
Oligonucleotide definition and use in PCR
Small synthetic primers
Define the boundaries of synthesis
Process of PCR
Heat denaturation (94 degrees) Primer annealling (55 degrees - 3'->5') Primer extension (72 degrees) Heat denaturation (94 degrees) Repeats itself
How are PCR products seperated
Gel electrophoresis
Analysis of PCR products
Determine presence/absence of product - allele-specific PCR
Determine product size by gel electrophoresis - oloigonucelotide ligation assay or direct sequencing
Connexin 26 delta35G assay
Most common inherited cause of deafness
Allele-specific mutation detection
Distinguishes between two alleles that may only differ by a single nucleotide
Designed so 3’ end base pairs with changed nucleotide but will not take place if they are not perfectly base-paired hence distinguishing between them
CFTR mutations are identified via
Cystic fibrosis genotyping assay.
Genotypes a panel of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic DNA isolated from blood
Difficulties in mutation analysis
Gene too big for PCR
Hard to deal with repetitive sequencing
GC-rich regions are difficult to PCR
Solution: southern blotting
DNA sequencing
Unknown mutations Ultimate method Complex and expensive Determines exact position of mutation Determines type of mutation Uses specific PCR product as template
Strand synthesis catalyst
DNA polymerase
Deoxynucleotides required for DNA sequencing
dATP, dCTP, dGTP, dTTP
BUT also: dideoxynucleotides: ddATP, ddCTP etc
What does it look like on sequencing gel
ddTTP, ddGTP lanes
Largest fragments at start (left)
Electric field goes from negative to positive
Exclude the end because of dye blobs - give messy sequence
Types of DNA sequencing
Traditional - 16 or 96 capillary
Clonal - 3.5-7GB of reads
What DNA is sequenced by clonal sequencing
PCR products
Long PCR product covering part or whole of a gene
Panels of selected genes known to be mutated for a particular phenotype
Very large ‘exome’ panels based on all coding genes
Clonal sequencing of gene panels (difficult)
You have bait of cRNA and your genomic DNA
This is target capture
You then use Magbead capture to select the bound oligonucleotides
Wash out the excess stuff
Digest RNA leading to enriched target
