Genetics Flashcards
What is human genetics?
•human genetics: the science of heredity and variation in humans
What is medical genetics?
•medical genetics: the subset of human genetics that is important in medicine and medical research
What is molecular genetics?
•molecular genetics: the study of the structure and function of individual genes
What is clinical genetics?
•clinical genetics: the application of genetics to diagnosis and patient care (in individuals and families)
Why are children referred to Clinical Genetics?
•Child
–Birth anomalies - malformations
–Dysmorphic features
–Learning difficulties
Why are adults referred to Clinical Genetics?
•Adult –Diagnosis –Predictive testing –Carrier testing –Family history (including cancer)
Why are pregnant women referred to Clinical Genetics?
•Pregnancy
–Known genetic disorder
–Abnormality detected on screening
–Fetal loss or recurrent miscarriages
How do you make a genetic diagnosis?
•Family tree –to detect a pattern of inheritance •Physical examination –to inform precise diagnosis –To direct testing •Genetic tests –Chromosomes (karyotype) Genes (DNA testing
What are common Non-genetic tests?
Non-genetic tests
Blood tests
•Enzyme assays–Inborn errors of metabolism
•Haematology–Thalassaemia
X Rays- Skeletal dysplasia: Achondroplasia
What are common genetic tests?
•Genetic tests Genomic architecture •Cytogenetics •Array-based techniques Gene faults •Sequencing •OLA assays MLPA tests
What are the 5 types of genetic tests?
•Diagnostic testing –Answer a specific question –Answer a broad question •Predictive testing –Must understand the implications •Carrier testing –Autosomal and X-linked recessive •Prenatal testing –Preventing genetic disease •Screening
What are the advantages of genetic testing?
–Early diagnosis •Early interventions •e.g. deafness –Carrier testing •Reproductive choices –Prenatal testing •Reproductive choices
What are the disadvantages of genetic testing?
–Do you want to know you’re going to get cancer sometime?
•Screening might help
–Alzheimer’s
•No treatment
–Will it affect your insurance prospects?
What is Genetic counselling?
An education process that seeks to assist affected (and/or ‘at risk’) individuals to understand the nature of the genetic disorder, the nature of its transmission and the options open to them in management and family planning.
Rare diseases – why bother?
•Individually rare •More than 7,000 known! •Add up to a lot of chronic disease •¼ to ⅓ of children in hospital •One of two areas of focus for “100kg” •Tell us a huge amount about biology –May inform therapy
What is Pharmacogenomics?
Analysing entire genomes, across groups of individuals, to identify the genetic factors influencing responses to a drug
What is Pharmacogenetics?
–Studying an individual’s genetic make up in order to predict responses to a drug and guide prescription
–Cancer
The study of inherited genetic differences in drug metabolic pathways which can affect an individuals response to drugs.
These differences may result in a positive response to a drug therapy or an adverse drug reaction.
Plays an important role in offering a stratified medicine approach to improve patient care.
How many nuclotides are in DNA?
•Almost all heritable information is written in DNA sequences
–3 × 109 nt
–750 megabytes uncompressed data
What is the structure of DNA?
This lack of a 2’ hydroxyl group (unlike RNA-transient-OH can bind to P on backbone) makes DNA stable
What direction is DNA written in
- DNA sequences are written in a 5´ → 3´ direction
- This is the direction in which DNA and RNA are synthesized
A DNA sequence may be written as:
5′-AACGTTCGGCCGGTAA
But usually, this actually means:
5′-AACGTTCGGCCGGTAA
TTGCAAGCCGGCCATT-5
•N.B. For a gene, the “sense” strand would usually be written (the one that ends up in the mRNA)
How long is DNA? How is it condensed?
DNA is 9cm it is packaged into a 9 µm chromosome
What are telomeres?
TTAGGG telomeric repeat sequence
Specialized replication machinery
-Telomerase, TERT
-Inactive in somatic cells
Telomeres shorten with somatic cell division
- Finite number of cell divisions to senescence
TERT reactivation in cancer
What is the mitochondrial genome?
Mitochondria are evolutionary remnants of endosymbiotic bacteria
Circular genome, Greatly reduced over time
16,569 bp
37 genes
Cytoplasmic inheritance: Oocyte, therefore only maternal inheritance
What are Diploidy and dosage?
•The chromosome complement is diploid –n = 23 –2n = 46 –DNA content (3×109 bp)×2 = 6×109 bp –Two copies of every gene (pat, mat)
•Dosage is important –For many individual genes •Haploinsufficiency –For all chromosomes •The exception: X chromosome The problem of dosage compensation
Vital statistics about the human genome?
•3 000 Mbp dsDNA per haploid genome –Chromosome 1 – 263 Mbp –Chromosome 22 – 39 Mbp •>90% is non-coding DNA •Approx. 20 000 protein-coding genes –Average gene size 50–100 kb –Average mRNA ~2 kb
•Single-copy sequences (non-repetitive) –Genes •Repetitive sequences –Interspersed repeats •e.g. Alu repeats –“Satellite” DNA •Large blocks of repetitive sequence •Heterochromatin
Coding genes: 20,769 Short non-coding genes: 9,079 Long non-coding genes: 13,564 Pseudogenes: 14,165 Gene transcripts: 195,565
What are genes?
•Functional units of DNA
–Genes are expressed
•Some place, some of the time
–Transcription – copying into RNA
–Translation – turning RNA into protein
•Not all
•Short and long non-coding RNAs inc. miRNAs
•Components
–Exons
–Introns
–Regulatory sequences
•Promoters, enhancers, locus control regionsWhat are gene families?
Several similar genes formed from one original gene
•Evolution of genes progresses by duplication and divergence
•Most genes belong to a family of structurally related genes
•Members of a gene family may be clustered or widely dispersed
•Pseudogenes
What are pseudogenes?
dysfunctional relatives of genes which have lost their protein codeing ability
OR
are no longer expresed-become inactivated but remain in the genome (can inerfere with lab tests)
What are Processed genes?
•Intronless copies of other genes –Usually remote from parent gene •Reverse transcription and reintegration –cf. retroviruses •Occasionally remain functional –e.g. PGK2 (testis-specific)
What is Repetitive DNA?
•Satellite DNA
–Large blocks of repetitive DNA sequence
•Interspersed repeats
–Scattered around the genome
What is Satellite DNA?
•Large blocks at centromeres and heterochromatic chromosomal regions •Simple tandemly repeated sequences –Many types e.g. alphoid DNA •Centromere repeat •Chromosome-specific •Size of blocks may be polymorphic –1, 9, 16, Y
Alphoid DNA: •A type of satellite DNA found at centromeres
•171-bp repeat unit
•Repeat unit sequence shows chromosome-specific sequence variation
–Probes for individual chromosome identification
•Alphoid DNA is required for assembly of the centromere- anchors Chr to spindle therefore controls Chr during division.
As it is Chr specific it can b used to identify individual human Chr’s
What is Alphoid DNA?
Alphoid DNA: •A type of satellite DNA found at centromeres
•171-bp repeat unit
•Repeat unit sequence shows chromosome-specific sequence variation
–Probes for individual chromosome identification
•Alphoid DNA is required for assembly of the centromere- anchors Chr to spindle therefore controls Chr during division.
As it is Chr specific it can b used to identify individual human Chr’s
What are Interspersed repeats?
•Scattered around the genome •Individual copies are present at many locations –Maybe between or within genes •Example: Alu repeat (a SINE) –Short interspersed nuclear element –500 000 copies, 300 bp, 5% of genome –Dispersed by retrotransposition Role in generation of molecular pathology
What are retrotranspoons?
Genetic elements that can amplify themselves in a genome. Alu is the most widespread class of retrotranspoons in the human genome
What is the aetiology of disease?
For any condition the balance of genetic and environmental factors can be represented by a point somewhere within the triangle.
•Achondroplasia – single gene but different heights
•Stroke – multifactorial with environment and polygenic factors – for some there may be single gene influence (Cadasil)
What are the 5 classifications of genetic disorders?
- Multifactorial / Complex: the interaction of multiple genes (genetic predisposition) in combination with environmental factors eg type II diabetes, ischemic heart disease.
- Single gene: a mutation in a single gene = Mendelian inheritance – AD, AR, XL, mitochondrial eg cystic fibrosis
- Chromosomal: an imbalance or rearrangement in chromosome structure eg aneuploidy, deletion, translocation
- Mitochondrial: a mutation in mitochondrial DNA
- Somatic mutations: mutation(s) within a gene(s) in a defined population of cells that results in disease eg breast cancer
What is Autosomal Dominant Inheritance?
- 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
- Applies to: Many diseases caused by gene mutations:
- Myotonic dystrophy
- Marfan Syndrome
- Huntington disease
- Chromosome deletions and duplications
- Chromosome deletion ie 22q11 deletion syndrome
What is penetrance?
Penetrance: The frequency with which a specific genotype is expressed by those individuals that possess it, usually given as a percentage.
•May alter with age eg Huntington disease by 80 years 100% penetrance penetrance
•Incomplete penetrance – not all relatives who inherit the mutation develop the disorder – eg BRCA1 mutations 80% life time chance of developing breast cancer
Percentage with a gene change who develop the condition
May be modified by other genetic variations
May be modified by environmental factors
What is Expressivity?
Expressivity: - variation in expression - the extent to which a heritable trait is manifested by an individual.
•Marfan Syndrome: aortic dilatation, lens dislocation, stretch marks
•BRCA1 mutation - +/- ovarian cancer, breast cancer
What is Anticipation?
Anticipation: - 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
What is a New dominant / de novo mutation?
A new mutation that has occurred during gametogenesis or in early embryonic development.
•The parents are not affected and the mutation is not detected in their blood cells. The child is the first to be affected in the family but can pass the mutation to their own children
What is Autosomal Recessive inheritance?
- Disease seen in one generation
- Does not tend to pass from one generation to next – parents usually unaffected
- Offspring of affected individual has low risk of disease – unless in consanguineous relationship
- Relatives may be asymptomatic carriers of the disease
- Affects males and females equally
- Gene mutations, not chromosomes
- Cystic fibrosis
- Many of the metabolic disorders
- Haemachromatosis
- Sickle cell disease
What is mitochondrial Inheritance?
•The sperm head does not have any mitochondria
•All our mitochondria are inherited from our mother
eg Maternally inherited diabetes and deafness
•Rare, males and females affected equally
•Only 27 genes within mitochondrial DNA
•Each cell has many mitochondria
•Every mitochondrium has many copies of each gene
•Mitochondria are inherited from the mother’s egg
•An affected mother will give all her children the mutation
•Highly variable expressivity and therefore severity of phenotype between relatives
•All the children of an affected man will be unaffected
What is X-linked (XL) inheritance?
- Males affected
- Females may be unaffected, mildly through to fully affected
- Males usually more severely affected than females
- Can NOT have male to male transmission
- Gene mutations and chromosome deletions/ duplications
- Duchenne Muscular dystrophy,
- Fragile X syndrome
- Red / green colour-blindness
- Haemophilia
Why can females have such a variable phenotype in X-linked inheritance?
•Most XL carrier females are asymptomatic or have mild symptoms, However they can have significant symptoms
•Two main factors influencing expression of phenotype
•X inactivation
•XL dominant vs XL recessive inheritance
Often can not predict a female phenotype accurately on prenatal testing
Why does X-inactivation cause a variable phenotype in females?
X-inactivation = Lyonisation: The process of random inactivation of one of the X chromosomes in cells with more than one X chromosome. Compensates for the presence of the double X gene dose.
•Is normal and occurs when there is 2 or more X chromosomes in a cell
•Occurs in early embryogenesis
•Random – which X is silenced
•Once inactivated an X-chromosome remains inactive throughout the lifetime of the cell and all its descendants
•Most, but not all genes switched off on the inactivated X
•Approximately 50% of cells express the normal gene
•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 – eg muscle in Duchenne Muscular dystrophy
What is XL dominant and XL recessive?
- XL dominant (rare)
- Rett syndrome (lethal in males, phenotype only in females)
- Fragile X syndrome – females:- asymptomatic to fully symptomatic ( due to X-inactivation pattern)
- XL recessive
- Red-green colour blindness
- Haemophilia
- Duchene Muscular dystrophy
- Carrier girls usually unaffected BUT can have significant symptoms because of X-inactivation (switches off normal X)
- Girls fully affected if inherit mutation from mum and dad
What is Family history taking? What symbols are used in FH trees?
- Helps make a diagnosis
- Helps clarify risk for our patient
- Helps us understand our patients view point
- Helps us identify who else may be at risk
- Helps us understand the patient’s support network
What are genome analysis methods?
- PCR
- DNA sequencing(Conventional/“Next-gen”)
- Array CGH
- Karyotyping
- FISH
Pathologies?
- Single gene variants (Known/Unknown gene)
- Copy number variants
- Chromosome number
- Structural changes
Red are “cytogenetics”
What is the hybridization principle?
Two DNA (or RNA or RNA+DNA) molecules will anneal (hybridize) into a duplex if and only if their sequences are complementary according to the Watson-Crick base-pairing rules.
Mismatched base-pairs destabilize the duplex and allow discrimination even between very similar targets.
e.g. mutant vs. normal
What is the Polymerase chain reaction?
- Amplification of DNA in vitro
- In vitro synthesis of large amounts of DNA by copying from small starting quantities
- Small synthetic primers (“oligonucleotides”) define the boundaries of synthesis
- DNA is synthesized by a DNA “polymerase” enzyme from the “monomers” (deoxy-ribonucleotides)
- Heat denaturation 94ºC
- Primer annealling 55ºC
- Primer extension 72ºC
The cycle then repeats. Defined end by primer in further cycles= uniform copies of original.
Exponential target region - doubles in every cycle - 230=109
Applications:
-Detect presence(Genomic sequence, Virus, Circulating fetal or tumour DNA)
-Generate template (Sequencing,Other analysis)
Max target ~500 bp One PCR product One sequence Only exons targeted Multiple gene sequencing not easy
What is Allele-specific mutation detection?
•Distinguishes two alleles that may only differ by a single nucleotide
•e.g. distinguish between a known disease-causing point mutation and the normal allele
•Only interrogates this one known mutation
•Example: OLA (oligonucleotide ligation assay)
–allele-specific oligonucleotides are designed so that their 3′ ends base-pair with the variable nucleotide
–they cannot be ligated if the 3′-end nucleotide is not perfectly base-paired, thereby distinguishing the two alleles
What is OLA (oligonucleotide ligation assay)?
–allele-specific oligonucleotides are designed so that their 3′ ends base-pair with the variable nucleotide
–they cannot be ligated if the 3′-end nucleotide is not perfectly base-paired, thereby distinguishing the two alleles
How do you hunt unknown mutations?
-If the identity of a disease-causing mutation is unknown, it must be searched for by sequencing the DNA of the region of interest
(One gene, Several genes, All the genes)
-The big trap – polymorphism
- There is a great deal of normal variation between genomes
- Pathogenic changes must be distinguished from harmless ones
Use ddATP terminator of ligation , ends in a specific base e.g:
A reaction contains:
dATP, dCTP, dTTP, ddATP
C reaction contains: dATP,dGTP,dTTP,ddCTP
labelled with colour so can sequence on computer
What is next-generation?
Individual molecues that are going to be sequenced are immobilised on a slidethe apply PCR like process into clonal clusters- each from a single molecule. Primed sequencing is done on each cluster. - ddCTP termination is reversicble so after each cycle the label is removed and another is added e.g ddATP and a different colour is added. 100,000 clusters per tile allowing you to read the sequence= decreased cost of sequencing whole genome
Modern NGS machines can sequence a whole human genome 30× over for ~£1000
600 million × 150-bp reads, ~100 GB data
Most of the data is not clinically interpretable
Introns etc, Only 2% exons
What is an exome?
Whole genome = 3000 Mbp “Exome” = 60 Mbp Smaller and more interesting “Clinical exome” (8000 genes) = 25 Mbp Cheaper (~£250)
Too many variants
10-20,000 protein-changing variations from the reference genome
Which one(s) are important?
Data analysis problem
Can you define a set of genes of interest?
Exome-based “diagnostic panels”
e.g. “muscular dystrophy genes”
“nephrotic syndrome genes”
Good for single-base changes
Not so good for large variants or copy-number analysis
How do you detect copy number variation (CNV)?
“Conventional” cytogenetics Metaphase chromosomes (karyotyping) Live cells Molecular cytogenetics -All cell-cycle stages -In situ (FISH- Metaphase/Interphase) -DNA (Array CGH/QF-PCR)
What is karyotyping?
Cell culture
G-banding
Variable resolution>5 Mbp
What are DNA-based methods for copy number? (4)
Array comparative genomic hybridization (aCGH)
Standard modern replacement for karyotyping- Whole genome copy number analysis
Cheap
Higher resolution
Whole-genome sequencing
Newer, cheap, take DNA smash it up in short chuncks of 50nt as these individual chucks are of known suquence they can be mapped out by a computer/aligned to correct chr position. Then can divide chr into 2 windows and count the number of read. as we should have 2 of each chr there should be the same number of reads in each window differences may suggest deletions
MLPA
Multiplex ligation-dependent probe amplification
Targeted method
Quantitative fluorescent PCR (QF-PCR)
Targeted method
Rapid, cheap
Prenatal applications
Why not always DNA?
DNA-based methods are good for dosage
Cheap, easier
Higher resolution
BUT they cannot detect genomic rearrangment e.f translocation as the overall nuber of Chr is the same therefore we need:
Cytogenetic analysis still needed for genome rearrangements
