Genetics Flashcards
1
Q
What is human genetics?
A
•human genetics: the science of heredity and variation in humans
2
Q
What is medical genetics?
A
•medical genetics: the subset of human genetics that is important in medicine and medical research
3
Q
What is molecular genetics?
A
•molecular genetics: the study of the structure and function of individual genes
4
Q
What is clinical genetics?
A
•clinical genetics: the application of genetics to diagnosis and patient care (in individuals and families)
5
Q
Why are children referred to Clinical Genetics?
A
•Child
–Birth anomalies - malformations
–Dysmorphic features
–Learning difficulties
6
Q
Why are adults referred to Clinical Genetics?
A
•Adult –Diagnosis –Predictive testing –Carrier testing –Family history (including cancer)
7
Q
Why are pregnant women referred to Clinical Genetics?
A
•Pregnancy
–Known genetic disorder
–Abnormality detected on screening
–Fetal loss or recurrent miscarriages
8
Q
How do you make a genetic diagnosis?
A
•Family tree –to detect a pattern of inheritance •Physical examination –to inform precise diagnosis –To direct testing •Genetic tests –Chromosomes (karyotype) Genes (DNA testing
9
Q
What are common Non-genetic tests?
A
Non-genetic tests
Blood tests
•Enzyme assays–Inborn errors of metabolism
•Haematology–Thalassaemia
X Rays- Skeletal dysplasia: Achondroplasia
10
Q
What are common genetic tests?
A
•Genetic tests Genomic architecture •Cytogenetics •Array-based techniques Gene faults •Sequencing •OLA assays MLPA tests
11
Q
What are the 5 types of genetic tests?
A
•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
12
Q
What are the advantages of genetic testing?
A
–Early diagnosis •Early interventions •e.g. deafness –Carrier testing •Reproductive choices –Prenatal testing •Reproductive choices
13
Q
What are the disadvantages of genetic testing?
A
–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?
14
Q
What is Genetic counselling?
A
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.
15
Q
Rare diseases – why bother?
A
•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
16
Q
What is Pharmacogenomics?
A
Analysing entire genomes, across groups of individuals, to identify the genetic factors influencing responses to a drug
17
Q
What is Pharmacogenetics?
A
–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.
18
Q
How many nuclotides are in DNA?
A
•Almost all heritable information is written in DNA sequences
–3 × 109 nt
–750 megabytes uncompressed data
19
Q
What is the structure of DNA?
A
This lack of a 2’ hydroxyl group (unlike RNA-transient-OH can bind to P on backbone) makes DNA stable
20
Q
What direction is DNA written in
A
- 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)
21
Q
How long is DNA? How is it condensed?
A
DNA is 9cm it is packaged into a 9 µm chromosome
22
Q
What are telomeres?
A
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
23
Q
What is the mitochondrial genome?
A
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
24
Q
What are Diploidy and dosage?
A
•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
25
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
```
26
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 regions
```
27
What 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
28
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)
29
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)
```
30
What is Repetitive DNA?
•Satellite DNA
–Large blocks of repetitive DNA sequence
•Interspersed repeats
–Scattered around the genome
31
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
32
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
33
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
```
34
What are retrotranspoons?
Genetic elements that can amplify themselves in a genome. Alu is the most widespread class of retrotranspoons in the human genome
35
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)
36
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
37
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
38
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
39
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
40
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
41
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
42
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
43
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
44
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
45
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
46
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
47
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
48
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
49
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”
50
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
51
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)
1. Heat denaturation 94ºC
2. Primer annealling 55ºC
3. 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
```
52
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
53
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
54
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
55
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
56
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
57
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)
```
58
What is karyotyping?
Cell culture
G-banding
Variable resolution>5 Mbp
59
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
60
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
61
What is cytogenetics?
Cytogenetics = study of chromosomes
•23 pairs of chromosomes per nucleus - 46,XY or 46,XX (diploid)
- gametes 23,X or 23,Y (haploid)
* 1-22 = autosomes
* X & Y = sex chromosomes
* Abormality – change in chromosome number &/or structure
62
Why study cytogenetics?
```
• 0.7% livebirths
• 5% stillbirths
• 50% miscarriages
• Up to 100% cancers
• Up to 40% of all conceptions!
• >140 known syndromes
Major contribution to congenital malformation & learning difficulties
```
63
What are copy number variations (CNVs)?
•“A DNA segment with a variable copy no. compared with a reference genome”
•Range 1Kb – several Mb
•12% of human genome
•One gene or contiguous genes
•Pathogenic or benign
•Familial or de novo
Most cytogenetic changes result in copy number variations
Types of CNVs
•Numerical (Aneuploidy/Polyploidy/Mosaicism)
•Structural (Deletion/Duplication)
64
How do cytogenetic abnormalities produce an abnormal phenotype?
* Dosage effect (deletion or duplication), for part of or a whole chromosome. Loss > deleterious than gain
* Disruption of a gene at a breakpoint/ inappropriate activation or inactivation of genes
* Position effect - gene in a new chromosomal environment functions inappropriately
* Unmasking of a recessive disorder
65
What are types of Numerical chromosome
| abnormality?
Diploidy = 2 copies of each chromosome
1. Aneuploidy = gain (trisomy) or loss (monosomy)
•Errors in meiosis/gametogenesis
• Maternal age - aneuploidy
• Paternal age - no significant risk
2. Polyploidy = gain whole sets (triploidy or tetraploidy)
3. Mosaicism = diploidy & aneuploidy
66
What is Non-disjunction?
Meiotic errors (Non-disjunction) - Failure of chromosome or chromatid separation
67
What are examples of autosomal aneuploidy?
Trisomy 21
Trisomy 18- Edwards
Trisomy 13- Patau's
all others are likely to cause death before birth
68
What are examples of sex chromosome aneuploidy?
* no age-related risk
* phenotype less severe than autosomal
* sexual orientation not affected
* Turner’s syndrome (45,X) - 1/2500
* Klinefelter’s syndrome (47,XXY) - 1/1000
* 47,XYY - 1/1000
* 47,XXX - 1/1000
69
What is Polyploidy?
* Errors at fertilisation
* Most usually triploidy
* 69,XXY or 69,XYY or 69,XXX
* 2% all pregnancies
* 99.9% spontaneously abort
* 1/57000 livebirths
70
What is Mosaicism?
Errors at early cleavage (mitotic non-disjunction)
```
Consequences of mosaicism
• Variable phenotype
• Variable lethality - foetal vs extraembryonic
• “Non-identical” identical twins
• Tissue-specificity – lateral asymmetry
• Recurrence risk (if gonadal)
```
71
Structural rearrangements & CNVs – deletion/duplication
* 1/2000
* Several genes
* Mostly sporadic
* Variable clinical expression – variable size of imbalance, other genetic and environmental effects
* Loss > deleterious than gain
72
What is a Microarray CGH (array CGH)?
* Genome-wide screen
* Hybridise sample & control DNA to a microarray “chip” – BACs or SNPs or oligonucleotides (1000s of DNA spots)
* Genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)
* Has replaced some karyotyping as 1st line test
Red bars = loss +0.3 shift of bars
Determining regions of potential copy number change
•At least 3 oligonucleotides required for any call
•Call imbalances >150000 bases, ie 150 Kb
•Database searches to ascertain pathogenicity of imbalances e.g. decipher
Clarification of imbalances
•Specific size of imbalance
•Trawl genomic databases to establish if pathogenic, and to ascertain gene content
•OMIM morbid genes or not – check against phenotype
•Parental studies – targeted arrays or FISH
•Supportive literature (Unique patient leaflets
Haploinsufficiency scores for genes in deleted region – low scores indicate greater likelihood of pathogenicity
73
Which patients qualify for array CGH?
* Moderate to severe learning & developmental disability (LDD) = 1-3% (IQ<70)
* Dysmorphic infants
* Pregnancies with abnormal ultrasound
* >40 genomic disorders
74
What are the advantages of array CGH?
* Early diagnosis -1st line test, reduces need for other tests and avoids the “diagnostic odyssey”
* High resolution = increased diagnostic hit rate
* Greater accuracy of location/size of imbalances
* Information on relevant genes
75
What are the disadvantages of array CGH?
* Dosage changes only – not balanced rearrangements or mutations
* Low level mosaics not detected
* Non-pathogenic & uncertain pathogenic changes detected
* Needs good quality DNA
* Future technologies – analyse mutations and dosage changes simultaneously, eg exomes, whole genomes
76
What is Quantitative fluorescent PCR (QF-PCR)?
* PCR amplification of short tandem repeats (STRs) [chromosome-specific, repeated DNA sequences] using fluorescent primers
* Products visualised & quantified as peak areas using an automated DNA sequencer
77
QF-PCR for Aneuploidy detection?
* DNA extraction from prenatal or blood sample
* PCR amplification – primers from chromosomes 13, 18, 21, X and Y
* DNA dosage in up to 4-5 markers/chromosome
* aneuploidy =>2 markers with abnormal dosage
78
Applications of molecular cytogenetics
* Detection of CMV’s
* Non invasive prenatal testing
* Pregnancy loss
* Pre-implantation studies
* Cancer
79
What is Non-invasive prenatal testing?
* Maternal blood sample
* Extract circulating free fetal DNA (<10% of total free circulating DNA)
* Assess aneuploidy of 13, 18, 21 (NGS)
* Risk for aneuploidy – invasive test to confirm
* Reduces no. of invasive tests
80
What do you do in a Spontaneous abortion?
• 50% chromosome abnormality
- Triploidy, 45,X, trisomy
•Tissues - skin, placenta, lung, cartilage, cord
•1. Macerate tissue
•2. Extract DNA - QF-PCR to assess aneuploidy; array CGH if normal
81
What is the link between cytogenetics and cancer?
```
Disease specific acquired chromosome changes
•Mostly translocations
•Several different acquired abnormal clones
•Diagnosis
•Prognosis
•Treatment (stratifiedmedicine)
-Leukaemia - bone marrow
-Solid tumour - tumour tissue
```
82
What is Reciprocal translocation?
Balanced Chromosome rearrangement
•break & exchange
• 1/500
• 5-10% phenotype risk
• reproductive risk- all may come together as normal but can also get the following:
83
What is Robertsonian translocation?
```
Balanced Chromosome rearrangement
• whole arm fusion
• acrocentrics
• 1/1000
• no phenotype risk
```
• reproductive risk
84
What is the future of cytogenetics?
* Array CGH as front-line test for wider cohort
* Free foetal DNA in maternal blood
* Introduction of next generation sequencing – sequence based analysis of chromosome changes
* Ultimate direction - move towards whole exome or whole genome testing
85
What is the effect of genetic bottlenecks?
They reduce diversity
86
What causes genetic bottlenecks?
Speciation
migraton
environment
disease
87
What is the two hit hypothesis?
Recessive at the cellular level (i.e. both copies of the gene inactivated to have effect).
Autosomal dominant pattern of inheritance of the cancer risk
88
What is the function of gatekeeper genes?
Gatekeepers – monitor and control cell division and death, preventing accumulation of mutations
directly regulate tumour growth: monitor and control cell division and death, preventing accumulation of mutations
89
What is the function of caretaker genes?
lCaretakers – improve genomic stability e.g. repair of mutations
90
What is the function of landscaper genes?
Landscapers – control the surrounding stromal environment
91
What are Tumour suppressor genes?
Protects cells from becoming cancerous
Loss of function increases the risk of cancer
e.g. APC, BRCA1/2, TP53, Rb
92
What are oncogenes?
* Regulate cell growth and differentiation
* Gain of function/activating mutations increase the risk of cancer
* e.g. Growth and signal transduction factors, RET gene
93
What type of inheritance pattern do most cancer syndromes show ?
autosomal dominant
94
Which cancers show autosomal recessive inheritance pattern?
* e.g. MUTYH associated polyposis, Fanconi anaemia, Ataxia telangiectasia
* Each parent is a carrier of one mutated copy, usually without the disease
* ¼ of children inherit both mutated copies and the cancer risk
* Appears to skip generations and may account for some sporadic cases
95
What are Sporadic Cancers?
Onset at older age
One cancer in individual
Unaffected family members
Cancers that are rarely genetic e.g. Cervix, lung
96
What are Familial Cancers?
Onset at younger age
Multiple primaries in individual
Other family members affected
Same type/genetically-related cancers
97
What is diagnostic genetic testing?
lInitial diagnostic testing (mutational analysis) usually performed on DNA from a relative affected with cancer to try to identify the familial mutation
98
What is predictive genetic testing?
If a mutation is identified in the family, predictive testing for the specific mutation may then be offered to other relatives to determine whether or not they are at risk
99
What is Retinoblastoma?
* Childhood ocular cancer
* Very rare: 1 in 15,000-30000 live births
* ~30-50 children/year in UK
* Classic example following Knudson 2-hit hypothesis
* Retinoblastoma (Rb1) gene
* Both genes mutated/lost in the tumour
* Genetic cases – one mutation is present in germline
* Inherited cases occur at younger average age
* Bilateral cases almost always germline
* 15% of apparently sporadic unilateral cases are germline (high new mutation rate)
* Other cancer risks e.g. osteosarcoma
* Early screening for children at risk
100
What is Familial Adenomatous Polyposis?
* Hundreds of bowel polyps (adenomas) from teens onwards
* Accounts for ~1% of bowel cancers
* High risk (up to 100%) of bowel cancer if untreated
* Other features – CHRPE, desmoid tumours, osteomas
* APC tumour suppressor gene
* Autosomal dominant inheritance
* Colonoscopies, total colectomy late teens/early 20s
101
What is Hereditary Non-Polyposis Colorectal Cancer?
AKA: NHPCC or Lynch syndrome
•Accounts for ~2-3% of bowel cancers
• Polyps are common, but not polyposis
• 60-80% risk of bowel adenomas or cancer from ~mid 20s onwards
• Other cancer risks e.g. endometrial/ovarian/stomach/GU
• Mismatch repair genes
• MLH1 (50%), MSH2 (40%), MSH6 (10%), PMS1/2 (rare)
• Autosomal dominant inheritance
102
What is the HNPCC – Amsterdam Criteria?
(All criteria must be met):
• One member diagnosed with colorectal cancer before age 50 years
• Two affected generations
• Three affected relatives, one of them a first-degree relative of the other two
• FAP should be excluded
• Tumours should be verified by pathologic examination
Patients with HNPCC should have colonoscopy ~every 18-24 months from age ~25
Removal of polyps/early detection of cancer improves survival
?Role of aspirin in preventing polyps
Prophylactic colectomy is not usually recommended
However, women may consider hysterectomy +/- BSO
103
What are the BRCA1 + 2 genes?
* BRCA1&2 are involved in DNA repair
* ~10% of cases of breast cancer under 40 and ~25% of those with strong FH
* Common mutations in Jewish and some other founder populations
* Autosomal dominant inheritance
* Risk of breast cancer 80%; ovarian BRCA1 – 40%; BRCA2 – 10-20%
* Some increased risk of other cancers – e.g. prostate, melanoma, male breast cancer
Options for BRCA1 & BRCA2 gene carriers
• Breast screening – annual MRI 30-50, annual mammography from 40 onwards
• Risk-reducing mastectomies +/- reconstruction
• Risk-reducing BSO (ovarian screening probably no use)
• Lifestyle changes
• Pharmacological prevention studies
* Mutations only responsible for 16% of familial cancers
* Account for 5-10% of all breast cancers
* Average woman has 12% risk of developing breast cancer (90 years)
* BRCA1 or BRCA2 increases risk of breast cancer to 85% (70 years)
* Increases risk of developing ovarian cancer from <2% to 55% (BRCA1) and 25% (BRCA2)
* Remove ovaries after family (reduces risk by 85%)
* Preventative mastectomy (reduces risk by 90%)
104
What is Li Fraumeni Syndrome?
* P53 mutations. Rare
* Autosomal dominant
* 50% risk of cancer by age 40, close to 100% lifetime
* Breast, sarcoma, brain, adrenocortical, leukaemia
* Avoid radiotherapy – risk of inducing cancers
* Limited screening - MRI for breast
* Poor prognosis
* Value of genetic testing less clear
105
What is LINE1?
•a Long Interspersed Nuclear Element
•100 000 copies, 6000 bp, 20% of human genome
•Promote their own mobility by retrotransposition
-ORF1, ORF2 gene products
-Small minority active
Role in generating single-gene pathology
Denono insertion of L1 can result in Haemophilia A
Unequal crossing over of both SINE and LINE can result in a pathology
106
What is SINE?
* Short interspersed nuclear element
* 500 000 copies, 300 bp, 5% of genome
* Primate-specific (unlike LINE1)
* Dispersed by retrotransposition therefore -dependent on LINE1-encoded proteins
* Both LINEs and SINEs have a role in generating single-gene pathology
107
What happens as a result of unequal crossing over?
•Recurrent large-scale duplications or deletions
-DiGeorge syndrome ~3-Mbp del 22q11.2
-Williams syndrome ~1.5-Mbp del 7q11.23
•Reciprocal del and dup may both be pathogenic
-17p11.2 1.5-Mbp segment
-Peripheral myelin protein 22 (PMP22) gene
•Duplication =HMSN1 (CMT1A)
•Deletion =HNPP (hereditary neuropathy with pressure palsies)
•Intragenic pathology
-Single-gene disorder e.g. Duchenne MD
-Dup and del both pathogenic
-Reading frame effects
108
What are Trinucleotide repeat expansions?
```
•Polyglutamine (polyQ) repeats (CAG)
–Neurodegenerative disorders
–Huntington’s disease
–Spinocerebellar ataxias
•Large non-coding repeat expansions
–Fragile X syndrome – transcriptional silencing
–Myotonic dystrophy
```
•Mutational instability
–Occasional (e.g. Huntington’s)
–Frequent (e.g. fragile X)
109
What is Huntington’s disease?
```
•Neurodegenerative disorder
–Involuntary movement
–Psychiatric changes
–Cognitive loss =dementia
–Neuronal loss
–Corpus striatum
–Cortex
•Autosomal dominant
–Heterozygous CAG repeat expansions
–Mutational instability
–Anticipation
```
•Progressive neurodegenerative disorder with motor, cognitive, and psychiatric disturbances - movements – memory – mood
•Movement disorder – chorea, dystonia, bradykinesia, swallowing/ choking, dysarthria
•Mood – depression, euphoria, apathy, anxiety, aggression, psychotic symptoms
•Cognition – loss of executive functioning, rigidity of thought, memory loss, dementia
•Mean age of onset is 35 to 44 years (range 2 - 80 years)
•Median survival time is 15 to 18 years after onset
•Autosomal dominant disorder
•Complete penetrance
•HTT gene at 4q16.3 (was IT15)
•Normal HTT gene contains, within exon 1, a run of CAG trinucleotide repeats
•The HD mutation = an expansion of CAG repeats ≥ 40 repeats
(a few people develop HD with CAG rpt of 36-39)
•Huntingtin protein - widely expressed in different tissues – function unknown.
•Abnormal protein – increased number of glutamine amino acids = polyglutamine (polyQ) expansion which alters protein structure and biochemical properties.
•PolyQ cellular protein aggregates form – ? disease causing
•Basal ganglia especially caudate nucleus primarily affected
110
What is Fragile X syndrome?
```
•One of the commoner single-gene causes of mental handicap
•X-linked
–“Semi-dominant”
–Males severely affected
–Large ears, testes
–Females less so, variably
•Peculiar inheritance
–Normal transmitting males
```
111
What is Dysmorphology?
* Morphology: the scientific study of the structure and form of either animals and plants or words and phrases
* Mainly features in face
* NB- features change with age; diagnosis often easier in children than babies/ adults
112
What are Congenital malformations?
```
•2-3% of births
•Single malformations often isolated events
•More likely to be genetic if:
-Multiple malformations
-Dysmorphic
-Family history of similar problems
```
113
What is the 22q11.2 deletion?
```
•Very variable-includes DiGeorge syndrome
•~1 in 5,000
•Learning difficulties ~70%
•Cleft palate ~15%
Velopharyngeal insufficiency 32%
•Congenital heart defect 75%
•Hypocalcaemia
•Seizures
•Immune deficiency
•Renal malformation
```
114
What is Achondroplasia?
Achondroplasia is a bone growth disorder that causes disproportionate dwarfism.
•~1 in 20,000
•Autosomal dominant- often new mutation
•Risk increases with paternal age
•Rhizomelic limb shortening
•Short stature
•Foramen magnum compression/ hydrocephalus
115
What is beckwith-Wiedemann syndrome?
```
overgrowth disorder
•~1 in 10,000
•Large tongue
•Ear pits/ creases
•Exomphalos
•Hemihypertrophy
•Neonatal hypoglycaemia
•Increased risk of Wilms tumour (nephroblastoma)
```
116
What is Down syndrome?
* Commonest chromosomal disorder
* ~1 in 800 live births
* Learning difficulties
* Congenital heart disease
* Hypotonia in neonates
* Single palmar cease
* Cataracts
* Hearing impairment
* Hypothyroidism
* Leukaemia
* Atlanto-axial instability
* Alzheimer's disease
117
What is Kabuki syndrome?
```
Kabuki syndrome is a rare, multisystem disorder characterized by multiple abnormalities including distinctive facial features
•~1 in 30,000
•Learning difficulties
•Congenital heart disease (50%)
•Poor growth
•Hearing impairment
•Cleft palate
•Premature breast development
Persistent fetal finger pads (96
```
118
What is mosaicism?
* Hypo- &/ or hyper-pigmented patches
* May follow Blaschko’s lines
* Diagnosis often requires skin biopsy
119
What is Peutz-Jeghers syndrome?
```
development of benign hamartomatous polyps in the gastrointestinal trac
•<1 in 50,000
•Gastrointestinal polyps
-Bleeding
- Obstruction
Malignancies:
•Colorectal
•Gastric
•Pancreatic
•Breast
•Ovarian
```
120
What is Treacher-Collins syndrome?
```
Treacher Collins syndrome is a condition that affects the development of bones and other tissues of the face
•~1 in 50,000
•Autosomal dominant
•Very variable
•Cleft palate
•Hearing impairment
```
121
What is Waardenburg syndrome?
```
Waardenburg syndrome is a group of genetic conditions that can cause hearing loss and changes in coloring (pigmentation)
•~1 in 250,000
•Sensorineural hearing impairment
•Iris heterochromia
•Premature greying
•White forelock
•Areas of skin hypopigmentation
•Congenital malformations (Hirschprungs/ VSD)
```
122
What is William’s syndrome?
```
•7q11 deletion
•~1 in 20,000
•Learning difficulties
•‘Cocktail party’ speech
•Congenital heart disease
-Supravalvular aortic stenosis
-Peripheral pulmonary artery stenosis
•Hypercalcaemia
```
123
What is Leber’s congenital amaurosis ?
•Leber’s congenital amaurosis (LCA)
•Rare inherited eye disorder
•Blindness at birth or in infancy
•Accounts for 10-18% of congenital blindness
•Over 22 genes implicated
•Recessive inheritance pattern
Trial with gene therapy in eye as Eye is immune privileged and accessible for subretinal injection
•RPE65(enzyme in retina) mutations identified in 1997 in LCA families
•In 1998, mutations found in naturally occurring blind dogs
Sub-retinal injection between RPE and photoreceptors with adeno-associated virus (AAV) containing human RPE65 and human RPE65 promoter- improvement in dog
•3 trials started in 2007 results published 2008, Phase one testing safety, UK trial injected 3 patients, Improvements in vision recorded in only 1 patient (the mildest)
124
What are Cytochrome P450 oxidases?
* Multigene family of enzymes found predominantly in the liver
* Responsible for the metabolic elimination of most drugs currently used
* Also important for converting pro-drugs to their active forms (eg codeine) codeine is metabolised to the analgesic morphine by CYP2D6, and the desired analgesic effect is not achieved in CYP2D6 poor metabolisers
125
What is the significance of CYP2D6?
* Highly polymorphic cytochrome 450 family member
* Metabolises 25% of drugs
* 6-10% of Caucasians are non-metabolisers (no active CYP2D6)
* 7% of Caucasians are ultra-rapid metabolisers (multiple copies of CYP2D6)
* CYP2D6 rate limiting step converting tamoxifen to its active metabolite endoxifen
* Poor metabolizers due to CYP2D6 polymorphisms are associated with worse survival
126
What is Non-invasive prenatal testing/diagnosis?
Cell-free DNA screening)
Maternal serum contains placental DNA which matches the foetus genome
NGS genome sequencing is performed on the cell-free DNA and the results can identify trisomy’s
Results are far more accurate than current methods (>99% compared to 85% for Downs) and have a much lower false positive rate (0.06% compared to 5.4% for Downs)
127
What is Prenatal diagnosis?
•Amniocentesis (~17 weeks)
•Chorionic villus sampling (~11 weeks)
•NIPD - New non-invasive method based on NGS of mothers blood (10 weeks).
NHS offer sex determination testing
Use for triploidy and single gene disorders testing (http://www.rapid.nhs.uk)
128
Why is Pharmacogenetics important?
* A UK study in 2004 found:
* 6.5% of UK hospital admissions related to adverse drug reactions (ADRs)
* Median hospital stay was 8 days
* This accounted for 4% of all hospital bed occupancy
* 2.3% of those admitted with ADRs died as a result
* Number of patients being under-treated due to genetic variation also likely to be high
* Most cancer drugs have response-rates of ~20% due to genetic variation in the tumour or patient. Many patients receive toxic treatments without benefit
129
What is Thiopurine methyltransferase?
* TPMT inactivates certain drugs
* Azathioprine (immunosuppressant used in organ transplantation and autoimmune disease)
* 6-mercaptopurine & 6-thioguanine (chemotherapies)
* TPMT gene polymorphisms reduce TPMT protein activity
* Severe toxicity if both copies of the gene have the variant
130
What is Ivacaftor?
* Cystic Fibrosis due to biallelic mutation of CFTR gene
* Ivacaftor therapy can significantly improve symptoms e.g. lung function, reduce sweat chloride concentrations, etc.
* Therapy enhances CFTR channel activity, i.e. increases probability of open channel.
* Significant improvement seen in patients with G551D genotype (hetero- or homozygous). Recommended for use
* No improvements seen in homozygous ΔF508del. Not recommended for use.
131
What is Succinylcholine?
* Related to the poison curare
* Muscle relaxant used in anaesthesia (to stop breathing)
* Usually wears off after a few minutes
* Rare BCHE gene variant homozygotes have reduced butyrylcholinesterase activity
* Effects may last for an hour or more and risk of death if artificial ventilation is not continued
132
What is Aminoglycoside?
* Mitochondrial MT-RNR1 gene encodes mitochondrial 12s rRNA
* G>A mutation at nucleotide position 1555 causes non-syndromic hearing loss (at later ages)
* Mutation changes the structure of the rRNA to resemble E-coli 16S rRNA
* Aminoglycosides more likely to bind to patients rRNA → increased risk of hearing loss at younger age
* Maternal inheritance
* Accounts for 30% of tendency to aminoglycoside toxicity
133
What is wafarin?
* Widely used oral anticoagulant to reduce embolism/thrombosis
* Decreases the availability of vitamin K
* Dose too low: Patient remains at risk
* Dose too high: Risk of haemorrhage
* Optimum warfarin dosage varies 20x between individuals
* In 2010, FDA recommended genotyping of CYP2C9 (one of the cytochrome p450 family) and vitamin K oxidoreductase complex-1 (VKORC1)
* These genes explain ~50% of genetic variability of warfarin activity
* Testing may reduce hospital admissions by 30%
134
What is Trastuzumab?
Herceptin
•20% of breast cancers have over-expression of HER2 (human epidermal growth factor receptor 2)
• These patients benefit significantly from trastuzumab – a monoclonal antibody to the HER2 receptor
135
What are BRAF inhibitors?
* Melanoma is notoriously resistant to chemotherapy treatment
* ~50% of melanomas have a somatic mutation in the BRAF gene
* A new targeted therapy Vemurafenib recently showed a 48% reponse rate compared with 5% for standard chemotherapy
136
What is Androgenesis?
Only paternal genes
137
What is Parthenogenesis?
Only maternal genes
| –Common in some other animals (insects, fish, reptiles) such as the mourning gecko
138
What is a Hydatidiform mole?
•Androgenetic
–Complete hydatidiform moles
–Mostly homozygous 46,XX
–Proliferation of abnormal trophoblast tissue
–Can develop into malignant trophoblastic tumour
–No (remaining) embryo
139
What is an Ovarian teratomas?
```
•Parthenogenetic conceptions
•Derived from oocytes which have completed first or both meiotic divisions
–Diploid
–Wide spectrum of tissues
–Predominantly epithelial
–No skeletal muscle
–No membranes/placenta
```
140
What are the consequences of uniparental conceptions in the mouse?
•Parthenogenetic embryos die due to failure of development of extraembryonic structures
–Trophoblast
–Yolk sac
•Androgenetic embryos die at 6 somite stage
–Well developed extra-embryonic membranes
–Poor embryo development
141
Why do uniparental conceptions fail?
```
•Different roles of maternal vs. paternal genes in determining developmental fate
•What is missing?
–Karyotype normal
–Gene dosage normal
•Concept of genomic imprinting
–Mothers and fathers somehow “imprint” their genes with a memory of their paternal or maternal origin
–How does this happen?
–What are the practical implications?
```
142
What is Genomic imprinting?
•A mechanism that ensures the functional non-equivalence of the maternal and paternal genomes
•Not encoded in the DNA nucleotide sequence
–i.e. epigenetic
•Depends on modifications to the genome laid down during gametogenesis
–Spermatogenesis vs. oogenesis
•Affects the expression of a small subset of 100-200 genes
–Evolutionarily conserved
Clinical consequences
143
What is Angelman syndrome?
```
•Facial dysmorphism
–Prognathism, wide mouth, drooling
–Smiling/laughing appearance
•Mental handicap
–Microcephaly
–Absent speech
•Seizure disorder
•Ataxic, jerky movements
Puppet children
•Deletion of chromosome 15- Found in both Angelman and Prader-Willi syndromes
•Always de novoRecurrence risks very low
Deletion in maternal Chr
```
144
What is Prader-Willi syndrome?
```
•Infantile hypotonia
–Feeding problems
–Gross motor delay
•Mental handicap
•Male hypogenitalism/
cryptorchidism
•Small hands and feet
•Hyperphagia
–Obesity
•Stereotypic behaviour
•Deletion of chromosome 15- Found in both Angelman and Prader-Willi syndromes
•Always de novo Recurrence risks very low
```
145
What determines which Chr is copied? (what genes are transcribed)
* Post-synthetic DNA modification
* Epigenetic –Does not normally alter DNA sequence
```
E.g: DNA methylation
•DNA methyltransferases
•Reversible
•Has to be “maintained” after replication
•Occurs at CG dinucleotides
–Many promoter regions spared
•CG “islands”
–Gene regulation
```
•Imprinted genes show monoallelic expression
–Epigenetic differences between maternal and paternal copy (allele)
–A “memory” of their distinct gametogenic histories
•Other chromatin structure differences between expressed and non-expressed allele
Other epigenetic mechanisms
146
What is Beckwith-Wiedemann syndrome?
```
•Fetal overgrowth
–High birthweight (>5 kg)
–+/- normal adult size
•Organomegaly
–Exomphalos
•Hypoglycaemia
•Asymmetry
•Tumour risk
•Sporadic occurrence
•(Epi)genetic abnormalities
–11p15
```
147
What is Russell-Silver syndrome?
```
•Growth retardation
–Fetal (IUGR)
–Persistent postnatal growth failure
•Triangular face
–Brain size more preserved
•Asymmetry
•Sporadic occurrence
```
148
What is Imprint “switching?
```
•Imprinting must be “remembered” during somatic development
•“Forgotten” before gametogenesis
•Erasure of grandparental imprint
•Establishment of new parental imprint
–During gametogenesis
–(Oogenesis for most genes)
```
149
What is X inactivation?
```
•By analogy with imprinted genes:
–In females, monoallelic expression is needed to ensure correct X gene dosage
–Achieved by epigenetic silencing
–Somatic cells remember silenced status
–Reversed in germ cells
X inactivation (Lyonization, 1961)
```
```
•Differences from imprinting:
–Whole X chromosome is silenced
–Random choice of parental chromosome
–Different in different cells
–Somatic cell clones “remember”
–Occurs early in embryogenesis
–occurs randomly in cells of early Blastocyst
```
150
What are the consequences of X inactivation?
•Females are “epigenetic mosaics”
–Composed of “patches” of cells, working on one or other X chromosome
–Carriers of X-linked mutations have some functionally defective and some normal cells
–The consequence of this “functional mosaicism” may be unpredictable, and may also be disease-specific
•Female carrier of an X-linked mutation
–Hypohidrotic ectodermal dysplasia
–Starch/iodine test
–Patches of skin with or without sweat glands
•Same principle for e.g. haemophilia, Duchenne muscular dystrophy
