Genetics part 1 Flashcards
What is genetic variation?
Describes the variation in the DNA sequence in each of our gene
What does genetic variation make us?
unique
What are the most common type of genetic variation amongst people?
single nucleotide polymorphism
What does each single nucleotide polymorphism represent?
Difference in a single DNA base
ACGT
in a person’s DNA
How much does SNP occur on average?
Once in every 300 bases
Also found in the DNA between genes
What does genetic variation result in?
Different forms, or alleles of genes
What does genetic variation explain?
Some differences in disease susceptibility
What can genetic basis to a disease be?
- Inherited
- Acquired
Different forms of inheritance
What is an example of acquired genetic changes?
Ovarian cancer
What is the advantage of finding genetic basis?
Define the disease
Define risk factors
characteristic or exposure of an individual that increases the likelihood of developing a disease or injury
Genetic molecular changes
Doesnt give us the disease but predisposes us to that disease
What is genetic testing?
Changes in chromosome, genes or protein
What does genetic testing do?
- confirms a suspected genetic condition
- Determine a person’s chance of developing or passing on a genetic disorder
- Determine whether a person is a carrier of a certain genetic mutation
What are examples of genetic testing?
- Diagnostic - looking at families
- Pre-syomptomatic
- Prenatal diagnosis
What is not always diagnostic?
phenotypic observation and test
What is mostly hidden?
Genetic risk factors
Identifying underling pathology
What is an example of inherited disease?
Sickle cell disease
Where can the prevalence of carrier frequency be very high?
West Africa
Who carries sickle cell disease gene?
1 in 20 people
What is progeria?
a rare syndrome in children characterized by physical symptoms suggestive of premature old age
What are rare diseases?
collectively common
What is factor V Leiden?
specific gene mutation that results in thrombophilia, which is an increased tendency to form abnormal blood clots that can block blood vessels
Define allele frequency
How common is the genetic variant within the population
What is some plausible explanations for the missing heritability
- Rare variants not captured in genotyping microarrays
- Many variants of small effect
- Structural variants not captured in short read sequencing
- Epistatic effects: non-linear gene-gene interactions
What is missing heritability a combination of?
- genes that supposedly underlie behavior genetic estimates of heritability simply do not exist
- Genetic effects are actually epigenetics
- Genetic effects are generally non-additive and due to complex interactions. a model has been introduced that takes into account epigenetic inheritance on the risk and recurrence risk of a complex disease
- Genetic effects are not due to the common SNPs examined in the candidate-gene studies & GWASes, but due to very rare mutations, copy-number variations, and other exotic kinds of genetic variants
- Traits are all misdiagnoses
- GWASes are unable to detect genes with moderate effects on phenotypes when those genes segregate at high frequencies
What does rare alleles cause?
Mendelian disease
What does low frequency give rise to?
Variants with intermediate effect
What are common variants implicated in?
Common disease by GWA
What is hard to identify by genetic means?
Rare variants of small effect
What does 5X increase risk of thrombosis give?
1 in 20 carrier frequency
What does 1.1x increase risk of coeliac give?
1 in 2.5 carrier frequency
When does an enzyme become dysfunctional?
one amino acid substitution to another
Cripple the catalytic site of enzyme
What can be tolerated?
Majority of the amino acid substitution
What are genetic markers?
Used to identify different features in DNA sequence that can be used to differentiate between individuals in a population
What does the genetic marker do?
Tag a piece of DNA and can be used to track genes in families or populations
What does genetic marker have?
Short DNA sequence such as a sequence surrounding a single base-pair change or a long one i.e. minisatellites
What is a short tandem repeat?
Short sequences of DNA (2-5 bp), repeated numerous of times in head-tail manner
Regions of non-coding DNA that contains repeat of same nucleotide sequence
What is genetic markers used for?
- Anonymous region of genome
- Fingerprints
- Fingerprinting in forensics
- Paternity testing
What is SNP?
- Polymorphic
2. When the frequency of the minor allele in the population is >1%
What is single nucleotide polymorphism?
- Single base pair difference in sequence of particular region of DNA from one individual compared to another of the same species or population
What does the little identifier serve as?
single nucleotide changes
What does the TERT gene provide?
Instructions for making one component of an enzyme called telomerase
Telomerase maintains structures called telomeres, which are composed of repeated segments of DNA found at the ends of the chromosome
SNP
In a population you can have either C or T in a population
The gene is in the TERT gene
What is the functional consequence synonymous?
Coding sequence of gene but does not change the amino acid
Effect: Low
What is Minor allele frequency?
The frequency at which the second most common allele occurs in a given population
What is MAF used in population genetics provide?
Information to differentiate between common and rare variants in the population
Rare variants (MAF < 0.05)
Appeared more frequently in coding regions than common variants (MAF >0.05) in this population
What does Global MAF illustrate?
11% of allele in the population are T
What is the least common genotype?
TT - Minor allele
C- Green
T- Red
Homozygous for C
Heterozygous for C/T
Homozygous for T - chromosome 2
What is disease gene identification?
Process by which scientists identify the mutant genotypes responsible for inherited genetic disorder
What does positional cloning start with?
Identification of candidate gene according to chromosomal location
Followed by mutation analysis in affected individuals
Positional candidate
- Identified through genome wide genetic linkage analysis and/or homozygosity mapping
Positional candidates
- Define candidate region – small region
- Obtain clones of all DNA in region
- Identify all genes in region
- Priotize them for mutation screening
- Test candidate gene for mutations in affected people
- Take patients with this disease and do genome wide genetic link analysis
- Genetic linkage analysis: establish linkage between genes
X-linked families
Males i.e.
- Haemophilia
- Mental retardation
What does dominant family pass
successive generations
Recessive disease
- Parents are asymptomatic (no symptoms) i.e.
- Sickle cell disease
- Cystic fibrosis
What is an example of dominant disease?
Huntington disease
What happens through consanguineous marriage?
The mutation becomes homozygous and causes autosomal recessive disease
What is SNP genotyping?
Measurement of genetic variations of SNP between members of a species
What does regions of extended homozygosity appear to contribute to?
Development of complex disease or traits involving recessive variants such as heart disease, hypertension and elevated total/low-density lipoprotein cholesterol levels
What is homozygosity mapping?
A common method for mapping recessive traits in consanguineous family
Why is homozygosity mapping powerful?
It does not require DNA of other family members than the affected offspring
What does the normal workflow of homozygosity mapping consist of?
Genome-wide linkage analysis with microsallelite or increasingly SNP
What does the homozygosity mapping minimize?
The need for sequencing multiple genes
What can you distinguish from homozygosity mapping?
Identification of a disease gene lines between 2 markers
- rs16954293
- rs9939133
What did sequencing all ~60 genes in the chromosome 16 region identify?
A homozygous loss of function mutations in the C16orf57
What is functional candidates?
Identified by a functional association with previously identified disease genes
What is Fanconi aneamia a result of?
Genetic defect in a cluster of proteins responsible for DNA repair via homologous recombination
Functional candidates
o Already found the disease gene
o Look at another patient that has the same disease but doesn’t have the mutation in the gene you found
o Fanconi anaemia – caused by bad genetic variants
o Fanconi pathway feed into the DNA repair proteins
o Fanconi anaemia – recessive
o BRCA1 famous risk factor – heterozygous
o Find one disease gene, investigate pathway, find other disease genes
What is exome sequencing?
Sequencing all of the protein-coding genes in a genome
- Select the subset of DNA that encodes proteins
- Sequence the exonic DNA using any high-throughput DNA sequencing technology
How many exons does humans have?
180,000 exons
constitutes about 1% of human genome
Approximately 30 million base pairs
What is the goal of exome sequencing?
Identify genetic variants that alter protein sequences at a much lower cost
Much easier to do
Cheaper
Computational space
Define a variant
Something in the sequence which is different from reference genome
How many exomes and genetic variants are there?
- Exomes - 23,000
2. Genetic Variants - 3 and 1/2 million genetic variants
What does allelic series describe?
Different mutant alleles of a gene that cause a range of phenotypewhereby each one carries a single point mutation within different regions of the same gene.
What are some examples of diagnosis?
- Confirmation, clarification
- Pre-symptomatic, monitoring, search for donor
- Pattern of inheritance, carrier testing
- Prenatal, preimplantation ,genetic counselling
What are examples of treatment?
- Tailor treatment: appropriate drugs
- Storing cells
- Rational drug design
- Gene therapy
What is wider pathology?
Mutation in patients with idiopathic disease
What is allelic series?
Identification of the gene that causes Freeman-Sheldon syndrome
What is RTEL 1 gene associated with?
Autosomal recessive dyskeratosis
What os MSH6 gene associated with?
Hereditary non-polyposis colon cancer type 5
What is DNA sequencing techniques?
- Sanger sequencing - DNA is copied many times - fragments of different lengths
What does DNA sequencing techniques determine?
many relatively small fragments of human DNA
What are the fragments aligned based on?
Overlapping portions to assemble the sequences of larger regions of DNA and entire chromosomes
What does the fluorescent ‘‘chain terminator’’ nucleotides mark?
The ends of fragments and allow the sequence to be determined
What are next generation sequencing?
techniques are new, large-scale approaches that increase the speed and reduce the cost of DNA sequencing
What are targeted gene panels?
Useful tools for analysing specific mutations in a given sample
What does the focused panels contain?
A select set of genes or gene region that have known or suspected association with the disease or phenotype
What does targeted gene panels produce?
Smaller, more manageable data set
make analysis easier
What is Sanger sequencing?
Still the gold standard to confirm results and for many genetic conditions with common variants e.g. thalassaemia
What are Targeted gene panels?
Based on a set of selected genes designed around a phenotype
What is whole exome/genome sequencing?
Open sequencing
Where the clinician and bioinformatic analysis defines the genes of interest
What are all the signal that has come out of the genome wide association?
Intergenic
What is missense (non-synonymous)?
Change one amino acid to another
What are indels?
Insertion or deletion of bases in the genome of an organism
Used as genetic markers in natural population
What are indels that are not multiple of 3?
Particularly uncommon in coding region but relatively common in non-coding regions
What is frame shift?
A genetic mutation caused by indels of a number of nucleotides in a DNA sequence that is not divisible by 3
How do you name a sequence variant?
C.1 = A of the ATG initiation codon
Specify coding sequence using unique identifiers of coding sequence NM
1601G>A cause factor V Leiden
NM = nucleotide sequence
Name the protein sequence
P.1 =The N-terminal Met residue
Use base number on human genome reference sequence
What is library preparation?>
- Take DNA of interest in patient of disease
- Shear DNA by sonication into fragments, 250bp
- Tie up the ends – phosphorylate/adenylate
- The adenylation/phosphorylation are crucial for the addition of linker/adaptor
- Library – all the fragments from one particular person
- Molecule is a simple oligonucleotide – fork and paired
- DNA insert and a lot of sequences put at the end – asymmetrical, tucked is a unique identifier
- Ligating adaptors onto a sheared DNA fragments
What is exon capture?
o Extract and sequence the exome in a genome and compare this variation across a sample of individual organisms
How to do exome?
- Make RNAse complementary all through the exons of every single gene
o RNA – bait – have biotin at the end
o Streptavidin bind biotin strongly
o Streptavidin – coating a magnetic bead
What is ILLumina dye sequencing
- Technique used to determine the series of base pairs in DNA (DNA sequencing)
- Primers attach to the forward strands and polymerase adds fluorescently tagged nucleotides to the DNA strand
- Only one base is added per round
- A reversible terminator is on every nucleotide to prevent multiple additions in one round
- Use four-colour chemistry, each of the four bases has a unique emission, and after each round, the machine records which base was added
- Once the DNA strand has been read, the strand that was just added is washed away
What is variant calling pipeline?
to allow researchers to rapidly identify and annotate variants
What does variant calling pipleline employ?
Genome Analysis Toolkit (GATK) – perform variant calling – a list of variants using genome viewer
What is the content of genome?
50 million base
How can you visualise genetic variant?
using genome viewer
What is Annovar?
annotate the genetic variants – predicting the likelihood of being pathogenic
What is ExAC?
Very big data base of nucleotides
What is variant calling pipeline?
Predict the severity of a variant on the function of a protein
What are the different criteria for variant calling pipeline?
computational data, functional and allele date to see it going from benign to pathogenic
What is the TERT protein?
1500 amino acids long
Predicted loss of function
Predicted to be null
What are the different ways of annotating single nucleotide variants?
- Differentiating
- Annotation
- Prediction
Differentiation
- Coding/Non-coding
- Known/unknown (dbSNP, ExAC, gnomAD)
- Homozygous/heterogous
Annotation
- Affected gene/transcript name
- silent, missense, nonsense, splicing, indel
- Amino acid change
- Loss of function versus hypomorphic versus neutral
Prediction
- Conservation, protein structure
2. SIFT, Polyphen, Mutation Taster, CADD score
What are approaches to variant classification?
- Searching medical literature
(can be decisive, clear known precedent) - Searching databases
- Using in-silico tools
What classes are useful for reporting variants?
Class 1 - Benign
Class 2 - Pathogenic
What mutation are in the oncogene?
- K-ras
2. DCC
What is De-novo mutation?
Genetic alteration that is present for the first time in one family member as a result of a variant
can be hereditary or somatic
Where does mutation occur?
In a person’s egg or sperm cell but is not present in any of the person’s other cells
What is acquired mutation?
– acquired early on in development or will be acquired in the germ cell
Some acquired diseases can be inherited to some extent
Who is closer to getting a colon cancer?
Individuals with inherited mutation in APC gene
What are monogenic diseases?
Single defective gene on the autosomes
Inherited according to Mendel’s Law
600 known
• 1 in every 200 birth
What can monogenic diseases be?
The mutation can be spontaneous and where there is no previous family history
What is an example of monogenic diseases?
- Cystic Fibrosis
2. Alzheimer’s disease
What is polygenic disease?
- A genetic disorder that is caused by the combined action of more than one gene
- Environmental factors and lifestyle factors come into play
- E.g. Alzheimer’s disease
What is Locus heterogeneity?
• Single disorder, trait, pattern of traits caused by mutations in genes at different chromosomal loci
What are examples of locus heterogeneity?
Dyskeratosis congenita
- Lots of phenotype in a lot of different body systems e.g. bone marrow
- It affects the health of stem cells
- Also found in skin and oral cavity
What can different genes lead to?
Same disease
What is penetrance?
Percentage of individuals who express gene as a particular phenotype
What does complete penetrance carry?
genes for a trait expressed in all the population and have clinical symptoms of the disease
What does incomplete penetrance demonstrate
individuals who acquire the disease do not express the trait, even though they carry the trait and accounts for only part of the population
What is variability in phenotypes caused by?
number of factors: modifier genes that can alter expression of alternative gene, environmental and genetic interactions
What is expressivity>?
When a phenotype is expressed to a different degree among individuals with the same genotype
• Either get variable penetrance or variable expressivity
• Example – Neurofibromatosis – NF1
• Some patients show
- Café-au-lait spots (moles)
- Neurofibroma tumours (tumours at the end of neurons)
Types of mutation
• Mutations in the coding sequence – change the amino acid sequence
• Epigenetics
- Study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself
• Genome quite heavily methylated
• Nucleosomes accessible to transcription factor and tightly packed
• Enhancers are a long way from gene
What doesn’t transcriptional control (quantity of proteins) necessarily change?
The coding sequence of genes but changes the amount of transcript produced
What are the causes of transcriptional control?
- Mutation in regulatory element i.e. enhancer element
- Repeat expansion
- Epigenetics
- Deletion/insertion/inversion/translocation
Translocation – Philadelphia chromosome
chromosome 9 and 22
Brings 2 genes together bcr and abl
Abl – tyrosine kinase – drives cell cycle
What are symptoms of single gene disorder?
- Sickle cell crisis
- Anaemia
- Infection
- Jaundice and gallstones
- Avascular necrosis
- Leg ulcer
- Delayed growth
What is the consequence if a particular polymorphic markers are close to disease mutation?
Inherited together
When is 50% of the diseases are going to be inherited together ?
If you have disease mutation on one chromosome, a polymorphic marker on the other chromosome
When is it always inherited with polymorphic marker?
If the disease mutation is right next to 3 repeats
What is X-linked recessive?
- Mother carries affected gene on X chromosome
- Females are carriers
- Only males are affected by the disorder
- Some example
- Duchenne Muscular Dystrophy
- Haemophilia
- Hunter disease
- Because men pass their Y chromosome on to their sons and their X chromosomes to their daughters, men who are affected will not pass the condition on to their sons but all their daughters will be carriers
What is X-linked dominant?
- Females usually have two X chromosomes, while most males have one X and one Y chromosome
- If a child has inherited the mutation from the X chromosome from one of their parents they will have the condition
- Woman with an X-linked dominant disorder has a 50% chance of having an affected daughter or son with each pregnancy
- The sons of a man with an X-linked dominant disorder will not be affected (since they inherit their only X chromosome from their mother), but his daughters will all inherit the condition
What is Autosomal recessive?
- Two of the defective genes are inherited
- Parents are carriers of the mutated gene
- The risk of an affected child being born is 25% for each pregnancy
- Examples: phenylketonuria, cystic fibrosis
What is autosomal dominant?
- Single copy of defective gene
- Conditions carried on the autosomes, males and females are equally affected
- Chance of it being passed on is 50% for each pregnancy
- If faulty gene is inherited, it will result in an affected individual
- Examples include: Huntington disease, polycystic kidney disease
What is imprinting genes?
epigenetic phenomenon that causes genes to be expressed in a parent-of-origin-specific manner
involves DNA methylation and histone methylation without altering the genetic sequence
These epigenetic marks are established (“imprinted”) in the germline (sperm or egg cells) of the parents and are maintained through mitotic cell divisions in the somatic cells of an organism
What is Angelman syndrome?
Neuro-genetic disorder - several different phenotypes and it caused by Ube3a gene
It is a ubiquitin ligase
Where is Angelman sundrome gene expresses from?
Maternally inherited chromosome
What did John Langdon state?
phenotypic features of down syndrome are the same regardless of ethnicity
What are the 4 phases of Mitosis?
Prophase
- Duplicated chromosomes are compacted
- Easily visualised as sister chromatids
- Chromosomes pair up
Metaphase
- Mitotic spindle latches onto the sister chromatids at the centromere
- Chromosomes are aligned in the middle of the cell
Anaphase
- The mitotic spindle contracts and pulls the sister chromatids apart
- Begin to move to opposite ends of the cell
Telophase
- The chromosomes reach either end of the cell
- The nuclear membrane forms again and the cell body splits into two (cytokinesis)
Where is Down syndrome caused by?
Non-disjunction
What is Non-disjunction?
- The failure of one or more pairs of homologous chromosomes or sister chromatids to separate normally during nuclear division
- Results in an abnormal distribution of chromosomes in the daughter nuclei
- It can happen in either meiosis I or II, sometimes in mitosis early on in development
Where does non-disjunction happen?
- Mainly maternal – 90% of the case – meiosis I error
- Paternal error – 8%
- Meiosis I – 3%
- Meiosis II – 5%
- After fertilization – 5%
What are Down syndrome phenotype features?
- 100% of down cohort – mental retardation
- Neuropathology seen in Alzheimer’s disease – plaques and tangles
- Muscle hypotonia – 100% of the cases
- Malformation to the bowel
- Increase in leukaemia – both acute lymphocytic and acute milocytic leukaemia
- Short stature
- Short broad hands
- Iris Brushfield spots
- Flattened facial profile and nose
What is mutated in people with down syndrome?
GATA1acute megakaryoblastic leukaemia
Where is GATA 1 located?
From X chromosomes
Patient with down syndrome?
- Mutation in GATA1 – frame shift mutation on exon 2
- Frame shift – shorter version of protein – translation from methionine 84 instead of methionine 1 – western blot
- Used specific antibodies to GATA1
Autosomal Recessive
• Cystic fibrosis
• Quite prevalent: 1 in 3000 new born, 1 in 25 people a carrier
• Affects different organ systems in body e.g. lungs and sweat glands
• Salty skin – diagnose cystic fibrosis
• Diagnosed even earlier through a neonatal blood spots
• Cause
- Mutation in cystic fibrosis transmembrane conductance regulator
• Large protein – 180,000 base pairs on chromosome 7 – long arm
• Encode 1500 amino acid proteins
• Takes chloride ions across membrane and down regulate sodium channel
• Salty skin – chloride channel affected – no reabsorption
• Lungs – changes in ions
• Sticky mucus – inflammation and bacterial infection
