Clinical Genetics Flashcards
PCR
Fundamental for any DNA application
PCR is used to amplify a specific region of DNA; primers flank the region you want to amplify.
Each cycle doubles the amount of DNA copies of your target sequence
Amplify enough DNA molecules so that we have sufficient material to sequence or for other applications
Fragment analysis
PCR based assay
PCR followed by capillary electrophoresis
Here we are sizing the PCR product
Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)
eg Huntington’s Disease
Example of a repeat Expansion disease
Huntington’s disease – severe neurodegenerative disorder
Caused by CAG repeat expansion in the Huntingtin (HTT) gene
Normal < 27 copies; Intermediate 27-35; Pathogenic > 35
Expanded protein is toxic and accumulates in neurons causing cell death
Diagnosed with fragment analysis
Sanger sequencing
Invented by Fred Sanger back in 1977
Cycle Sequencing; based on the same principles as PCR
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
Up to 800 bp of sequence per reaction
Accurate (99.99%)
One reaction = one sequence
Slow and low-throughput
Costly to perform ££££
Here we are reading the dyes to obtain the DNA sequence
We can identify single nucleotide polymorphisms (SNPs), or mutations in this way
Detection of a mutation in a family by use of Sanger Sequencing
R1042G mutation in gene C3 segregates with affected individuals
Mutation causes disease cutaneous vasculitis
FISH
Fluorescent in situ hybridisation 1969, Gall & Pardue Cultured cells, metaphase spread Microscopic (5-10Mb) To detect large chromosomal abnormalities
Extra chromosomes
Large deleted segments
Translocations
More to follow in your next two lectures…. Chromosome abnormalities
Design Fluorescent probe to chromosomal region of interest
Denature probe and target DNA
Mix probe and target DNA (hybridisation)
Probe binds to target
Target fluoresces or lights up
Array CGH
Array comparative genomic hybridisation
For detection of sub-microscopic chromosomal abnormalities
Patient DNA labelled Green
Control DNA labelled RedPatient array comparative genomic hybridisation profile
Increased green signal over a chromosomal segment in the patient DNA
Indicates a gain in the patient sample not present in the parents
mlpa
Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets with only a single primer pair.
Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA.
We use MLPA to detect abnormal copy numbers at specific chromosomal locations
MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions.
One probe oligonucleotide contains the sequence recognized by the forward primer, the other contains the sequence recognized by the reverse primer.
Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe.
Perform fragment analysis of MLPA product
An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations
For example, probes may be designed to target various regions of chromosome of a human cell
The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome.
Next Generation Sequencing
Next Generation Sequencing has replaced Sanger sequencing for almost all sequencing tests in the labTechnological advances since the end of the human genome project
Decrease in the cost of DNA sequencing
Since the end of 2007, the cost has dropped at a rate faster than that of Moore’s law Development of new NGS methods began 13 years ago with 454 pyrosequencing
DNA sequencing throughput jumped 10 orders of magnitude
Solexa sequencing-by-synthesis (SBS) developed end of 2005
Sequencing market is now dominated by Illumina SBS sequencing.
An end to sequential testing
Wider range of tests in a shorter time for less money
Current strategy: Disease panels
Enriching to sequence only the known disease genes relevant to the phenotype
Panels expandable to include new genes as they are published
Potentially pathogenic variants confirmed by Sanger sequencing
Exome sequencing
There are ~21,000 genes in the human genome
Often we are only interested in the gene protein coding exons or ‘exome’ represents 1-2% of the genome
Some ~80% pathogenic mutations are protein coding
More efficient to only sequence the bits we are interested in, rather than the entire genome
Costs £1,000 for a genome, but only £200-£300 for an exome.
Target enrichment
Capture target regions of interest with baits, then incubate dna from patient with RNA baits. Rna baits are complementary to genes. Form hybridisation step. Capture fragments that have been hybridised. Wash away bits of fna not interested in.
Potential to capture several Mb genomic regions (typically 30-60 Mb
Genome sequencing
Universally accepted that genome sequencing will become commonplace in diagnostic genetics
Not all tests will automatically move to whole exome sequencing / whole genome sequencing
Panels/single gene tests may still be more suitable for some diseases.
Capillary-based methods: Repeat expansions, MLPA, Family mutation Sanger sequencing
Array-CGH: large sized chromosomal aberrations
Exome and Genome sequencing
Result interpretation is the greatest challenge
20,000 variants per coding genes ‘exome’
Need for good variant databases of well phenotyped cases/control sequences
Ethical considerations
Modified patient consent process
Data analysis pathways – inspect relevant genes first
Strategy for reporting ‘incidental’ findings
Infrastructure and training (particularly IT)
The NHS diagonostic laboratory
Accredited laboratory: ISO standard 15189 for Medical Laboratories.
Scientific, technical and administrative staff.
Provide clinical and laboratory diagnosis for inherited disorders.
Liaise with clinicians, nurses and other health professionals
Provide genetic advice for sample referrals and results
Carry out translational research for patient benefit
Train clinical research fellows, clinical scientists, post- and undergraduate students.
The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and management.
Perform specific tests with proven:
Clinical Validity: How well the test predicts the phenotype
Clinical Utility: How the test adds to the management of the patient
UKGTN (UK genetic testing network)-approved tests
In-depth and up-to-date knowledge of the genetic diseases covered
Translational Research Diagnostic Diagnosis Management and Treatment Inform clinical trials
Family mutation
Diagnosis
Interpretation of pathogenicity
Predictive
Life choices, management
risk of child getting disease. This is under strict genetic counselling.
Carrier (recessive)
Life choices, management
Diagnostic testing is available for all Consultant referrals
Clinical Geneticists most common referrers
Informed consent
Genetic counselling
Implications for other family members
Diagnostic testing
All referrals via Regional Genetics Centres
Close liaison with nurse specialists, genetic counsellors, clinicians during testing
International guidelines for predictive testing
Follow up at clinics, nurse led clinics, nurse telephone clinics as requiredPathogenic mutation
Normal variation
Polymorphism
Novel variant
Investigations to establish significance
Previously published/detected variant of uncertain significance…
Interpreting results
How to establish if a mutation is pathogenic?
ACMG Guidelines (Richards et al. 2015 Genet Med. 17:405-24)
Reported in ExAC (dbSNP / 1000 Genomes / NHLBI)? How common? Look at sub-populations Previously published: Read the papers! Segregation Controls (which population?) Functional studies
Transcript analysis
Is the gene expressed in blood / fibroblasts?
In-vitro splicing experiments
Functional studies
E.g. Ion channel function in Xenopus oocytes
Do not report known polymorphisms
Conservative approach to reporting novel mutations of uncertain pathogenicity
‘Uncertain significance’
‘Likely to be pathogenic’
Request samples from family members
? Continue testing other genes ?
Discuss results with consultant in person, and/or with local expert consultantDo not report known polymorphisms
Conservative approach to reporting novel mutations of uncertain pathogenicity
‘Uncertain significance’
‘Likely to be pathogenic’
Request samples from family members
? Continue testing other genes ?
Discuss results with consultant in person, and/or with local expert consultant
The 100,000 Genomes project
100,000 genomes project
Bring direct benefit of genetics to patients
Enable new scientific discovery and medical insights
Create an ethical and transparent programme based on consent and patient engagement
To kick-start the development of a UK genomics industry
Personalised medicine.
UK – wide collection
GMCs (genomic medicine centres)
Who/what is being sequenced?
Rare diseases - families
Cancer – germline and tumour samples
Clinical Interpretation
Genomics England Panel App
Community driven genetic interpretation
Crowdfunding research
‘Experts’ develop lists of possible genes than can cause a disease phenotype
These panels are reviewed by the community
Diseases have specific sets of virtual gene panels as a first port of call to look for pathogenic mutations
Thus we can focus on specific regions of the patients genome we think are important.
Classification of variants by genomics England
Maximise diagnostic efficiency
Balance sensitivity and specificity
Variants within virtual panel divided into three tiers
Expert review is required
Tier 1 variants
Known pathogenic
Protein truncating
Tier 2 variants Protein altering (missense) Intronic (splice site)
Tier 3 variants
Loss-of-function variants in genes not on the disease gene panel
What is a nucleosome
Nucleosome is fundamental unit of DNA – eight histones and two turns of DNA
Describe the structure of chromosomes
Chromosomes usually exists as chromatin
DNA double helix bounds to histones
Octamer of histones form nucleosome
Euchromatin
Extended state, dispersed through nucleus
Allows gene expression
Heterochormatin
Highly condensed, genes not expressed
Humans have 23 pairs of chromosomes 22 pairs autosomes, 1 pair sex chromosomes XX or XY Metacentric p & q arms even length 1-3, 16-18 Submetacentric p arm shorter than q 4-12, 19-20, X Acrocentric Long q, small p p contains no unique DNA 13-15, 21-22, Y Humans have 23 pairs of chromosomes 22 pairs autosomes, 1 pair sex chromosomes XX or XY Metacentric p & q arms even length 1-3, 16-18 Submetacentric p arm shorter than q 4-12, 19-20, X Acrocentric Long q, small p p contains no unique DNA 13-15, 21-22, Y
Describe the process of the cell cycle
Interphase
G1 = Cell makes a variety of proteins needed for DNA replication
S = synthesis; chromosomes are replicated so that each chromosome now consists of two sister, identical chromatids
G2 – synthesis of proteins especially microtubules
Some cells don’t replicate; some are senescent.
Not interphase:
Mitosis, cytokinesis.
G0 phase is for cell cycle arrest.
Examples of chromosomal changes
Numerical:
-can detect through traditional karyotyping, FISH, QF-PCR, NGS
Structural:
-can detect through traditional karyotyping, FISH.
These techniques are good for visualising big changes to the genome – and those big changes fall into these two categories.
Numerical and structural.
Can you give me an example of a numerical change? Down’s
What’s the genetic change which causes Down’s syndrome? Trisomy 21
Define Numerical abnormalities
HAPLOID:
one set of chromosomes (n=23) as in a normal gamete.
DIPLOID:
cell contains two sets of chromosomes (2n=46; normal in human)
POLYPLOID:
multiple of the haploid number (e.g. 4n=92)
ANEUPLOID:
chromosome number which is not an exact multiple of haploid number - due to extra or missing chromosome(s) (e.g. 2n+1=47)
What is disjunction
Pulling apart at anaphase.
Through what process does aneuploidy arise?
Non disjunction.
What is mosaicism
The presence of two or more genetically different cell lines derived from a single zygote.
Mechanisms of mosaicism
2 mechanisms:
Post-zygotic nondisjunction, i.e. mitotic non-disjunction = All 2n to mixture of 2n and 2n+1
Anaphase lag, i.e. trisomic rescue = All 2n+1 to mixture of 2n+1 and 2n
What is anaphase lag
Anaphase lag describes a delayed movement during anaphase, where one homologous chromosome in meiosis or one chromatid in mitosis fails to connect to the spindle apparatus, or is tardily drawn to its pole and fails to be included in the reforming nucleus. Instead, the chromosome forms a micronucleus in the cytoplasm and is lost from the cell.[1] The lagging chromosome is not incorporated into the nucleus of one of the daughter cells, resulting in one normal daughter cell and one with monosomy.[2] Anaphase lag is one of several causes of aneuploidy and one of several causes of mosaicism. Anaphase lag can also cause a rescue of the daughter cell if the cell was originally trisomy.[1]
Monosomy
Autosomal are very very rare, found one case report from 1967
Relatively common sex chromosome monosomy = Turner’s
Full monosomy arise by NDJ
Partial monosomy (microdeletion syndromes) far more common – mechanism different
How does Turner’s 45, X arise?
Nullisomic gametes fertilised with a sperm carrying an X chromosome will be XO (Turners-physically female).
Nullisomic gametes fertilised with a sperm carrying a Y chromosome will be YO-lethal
What causes triple X syndrome, Klinefelter’s syndrome? (genotype)
Disomic gametes XX \+ X chr = XXX = triple X syndrome \+ Y chr = XXY = Klinefelter’s (physically male) XY \+ X chr = XXY = Klinefelter’s \+Y chr = XYY = XYY syndrome
Describe the types of numerical abnormalities
Types (all can be mosaic) Autosomal Trisomy 13, 18, 21 Sex chromosomes XO, XXY, XYY
Mechanism
Nondisjunction
Anaphase lag
How can we do prenatal diagnosis of infants for Turners
Chorionic Villus sampling
11-14 weeks
Miscarriage rate 0.5% to 1%
maternal contamination
transverse limb defects
Amniocentesis >16 weeks extraction of amniotic fluid Biochemical diagnosis possible miscarriage risk (0.5-1%)
What is G-banding
Giemsa stain Metaphase Line-up based on Size Banding Centromere position
Giemsa highlights heterochromatic regions which are less likely to contain genes. But the crucial thing is that the banding can be used to differentiate between chromosomes ant to compare chromosomes.
Describe chromosome banding
Most common = G-banding
G = Giemsa
Why bands?
Chromatin
2 different sorts: euchromatin & heterochromatin
Euchromatin = GC-rich; loosely packed; genes active
Heterochromatin = AT-rich; tightly packed; genes inactive
Stain differently
Describe FISH
Fluorescent in situ hybridisation Cultured cells, metaphase spread Microscopic (5-10Mb) Fluorescent probe Denature probe and target DNA Mix probe and target DNA Probe binds to target -because it uses cultured cells, this takes longer than QF-PCR
Describe prenatal diagnosis i.e foetal testing
Invasive
Amniocentesis (14-20 wks, amniotic fluid)
Chorionic villus sampling (CVS) (11-14 wks, placental cells)
Non-invasive
Cell free foetal DNA (cffDNA): DNA fragments in maternal plasma (10 wks onwards)
Actually for trisomies still need confirmation with amnio/CVS
Down syndrome-clinical features and info
1 in 650-1000 live births Most common cause of mental retardation Hypotonia, particularly in newborn period Developmental delay Cardiac abnormalities GI abnormalities Acute Lymphocytic Leukaemia/Acute Myeloid Leukaemia – 10-20 x relative risk Conductive hearing loss Features of Alzheimer’s >40 years.
95% patients non-disjunction (usually maternal meiosis I)
5% Robertsonian translocation involving chromosome 21
~2% mosaic (ie only some cells contain an extra chromosome)
‘Older Egg Model’ – maternal age effect
Patau syndrome
~1 in 10 000 live births Midline defects Hypotelorism Holoprosencephaly Midline cleft lip/palate Scalp defects Post axial polydactyly Heart defects/renal abnormalities Survival – most die by 1 month Hypotelorism=abnormally close eyes Holoprosencephaly – forebrain doesn’t divide properly-causes facial malforamtions as well Post-axial=lateral side of foot or hand (little finger side)
Edwards syndrome
Incidence ~1 in 6000 live births Intrauterine growth retardation Micrognathia (small lower jaw) Cleft lip +/- palate Short palpebral fissures (gap between eye lids) Fixed flexion deformities of fingers Heart defect >95% Inguinal/diaphragmatic herniae Renal malformations Survival 30% die by 1 month 50% die by 2 months 90% by 1 year
Turner syndrome
Incidence 1/4000 female births 45X0 or mosaic (45X0/46XX) Raised nuchal translucency At birth: oedema of hands and feet Neck webbing ?coarctation of aorta ?renal malformation Short stature Infertility secondary to gonadal dysgenesis Intellectually normal Nuchal translucency is a collection of fluid under the skin at the back of your baby's neck. A cystic hygroma is a collection fluid-filledsacs known as cysts that result from amalformation in the lymphatic system. Coarctation = narrowing
Klinefelter
(47,XXXY)
Incidence 1/1000 male live births
Phenotype mild and variable – some cases undetected
Barr body present
NDJ paternal meiosis I (50%), others NDJ maternal meiosis or zygotic mitotic error (mosaic)
Variants: 48,XXYY, 48,XXXY etc
May present prenatally, during childhood with behavioural problems, or adulthood with infertility
Tall stature
Eunachoid body habitus
Some behavioural and minor learning difficulties
Lack of secondary sexual characteristics – treat with testosterone
Infertility
