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
Structural abnormalities of chromosomes
Translocations Reciprocal Robertsonian Inversion Deletion Duplication Rings Isochromosomes Microdeletions/Microduplications
How do structural abnormalities of chromosomes arise?
Double strand DNA breaks
Occur throughout cell cycle
Generally repaired through DNA repair pathways
Mis-repair leads to structural abnormalities
What are reciprocal translocations
Exchange of two segments between non-homologous chromosomes
So mechanism is called Non-Homologous End Joining (NHEJ)
What are translocations due to inappropriate Non homologous end joining
Translocation is where we happen to have two double strands breaks, each on a different chromosome.
There are DNA mechanisms within the cell which monitor genome integrity and when they detect a fault, will repair that fault.
However, what happens very occasionally is that instead of joining together the correct two bits, the DNA repair mechanism happens to stitch together the chromosome in incorrect pairs
What we see therefore is most of one chromosome, with the end of another chromosome attached and vice verse in this chromosome
The “der” here stands for derivative, meaning “the result”
The DNA repair mechanism is called “non-homologous end joining”: end joining because it’s joining together two ends and non-homologous because it’s irrespective of the DNA sequence joined together
These are also known as balanced translocations
It’s thought that they form spontaneously during meiosis
The key characteristic is that there is no net gain or loss of genetic material – it’s all there, just in a different place.
They can involve any chromosome and the fragments can be of any size
They are relatively common – estimates suggest that they occur in 1 in 930 people
What is the difference between balanced vs unbalanced chromosomal translocations
Balanced = have the right amount of each chromosome just maybe not in the expected place!
Unbalanced = too much or too little of a particular chromosome
What balanced translocation affects carriers
Philaldelphia chromosome -abnormal chromosome 22
Leads to Chronic myeloid leukaemia (CML)
BCR=breakpoint cluster region (Function of normal protein product not known)
ABL=protooncogene
Fusion of genes leads to an activated oncogene
What are reciprocal translocations
Exchange of two segments between non-homologous chromosomes
Balanced translocation – no net gain or loss of material
Usually no deleterious phenotype unless breakpoint affects regulation of a gene
Carrier of balanced translocation at risk of producing unbalanced offspring
Unbalanced individuals at significant risk of chromosomal disorder
What are the consequences of reciprocal translocations in meiosis
A reciprocal translocation means that there is no loss or gain of material and so there’s often little consequence to the cell of carrying a reciprocal translocation.
However – that changes when we look at what happens to these chromosomes during meiosis.
Here we have a pair of chromosomes, chromosomes 11 and 22 and we have a reciprocal translocation at this point here.
In meiosis, you might be lucky in that the way the chromosomes separate is like this or this – where the correct amount of each chromosome goes into the resultant cell.
However, if we think about how these chromosomes pair up before separating, we find that they form this structure called a pachytene quadrivalent
What can happen is that the chromosomes separate along this horizontal blue line, resulting in one cell having a gain in yellow chromosome and a loss of the end of the purple chromosome; the other daughter cell has a loss of the end of the yellow chromosome and gain of the purple chromosome.
Alternatively, the chromosomes could separate along this vertical blue line.
Again, this will result in an unbalance arrangement where, in each daughter cell, there is loss of one end of a chromosome and gain of the end of the other chromosome.
The exact consequences of inheriting a unbalanced rearrangement depend on the particular chromosomes involved and the size of the translocated material.
What is the clinical result of unbalanced reciprocal translocation?
Many lead to miscarriage (hence why a woman with a high number of unexplained miscarriages should be screened for a balanced translocation)
Learning difficulties, physical disabilities
Tend to be specific to each individual so exact risks and clinical features vary
What are Robertsonian translocations?
When two acrocentric chromosomes break at or near their centromeres, when the fragments are joined together again it’s possible for just the two sets of long arms to be brought together and there’s loss of the satellites.
This is called a Robertsonian translocation.
If this happens in a cell, how many chromosomes will be have? 45
The only genetic material we’ve lost are these satellites and the cell can do without those and so this isn’t a problem for the cell.
For now, let’s move on to another type of translocation – the Robertsonian translocation.
This is named after American cytogeneticist who first described them
Only affect acrocentric chromosomes – ie. Those which have the centromere near the chromosome tip. These are chromosomes 13, 14, 15, 21 & 22
Most common Robertsonian translocation involves chromosomes 13 and 14, which accounts for approximately 1/3 of all Robertsonian translocations
Results in loss of two short arms and fusion of the two long arms, with either one or two centromeres
The resultant chromosome usually contains the long arms of different chromosomes (unusual to see, for example, the maternal and paternal long arms of chromosome 13 fused together).
Two acrocentric chromosomes join near centromere with the loss of p arms
Balanced carrier has 45 chromosomes
If 46 chromosomes present including Robertsonian then must be unbalanced
p arms encode rRNA (multiple copies so not deleterious to lose some)
Robertsonian translocations 13;14 and 14;21 relatively common. 21;21 translocation leads to 100% risk of Down syndrome in fetus
What are the terms given to chromosomes with diff centromere positions?
Centromere:
It’s not always in the middle.
There are terms given to chromosomes depending on where the centromere is: metacentric if it’s in the middle, submetacentric if it’s displaced from the middle and acrocentric if it’s essentially at the end of the chromosome, such that the p arm is just this little stubby satellite structure.
What are the consequences of Robertsonian translocations
In this example, we’re looking at a robertsonian translocation between chromosomes 14 and 21.
Again, let’s consider what happens to these chromosomes during meiosis
It could be that the daughter cell ends up with the normal copy of chromosome 14 and 21 – in which case, this gamete can go on to form a normal child after fertilisation.
Or, perhaps the daughter cell just has the translocated chromosome. Again, this gamete is capable of forming a normal child.
However, it could be that when the chromosomes segregate, the daughter cell ends up containing the normal chromosome 21 plus the translocated chromosome. After fertilisation, these will be joined by another chromosome 14 and a chromosome 21 – resulting in a normal number of chromosomes 14, but triploidy of chromosome 21. As you know, this will result in Down’s syndrome.
There are three other ways in which these chromosomes can segregate but these will either result in monosomy of one or the chromosomes or trisomy 14 – all of which are incompatible with life.
Remember, carriers of Robertsonian translocations can be phenotypically normal and it is possible for them to have a child with a normal chromosomal complement, or even a normal carrier of the same Robertsonian chromosome.
However, couples where one partner is a carrier of a Robertsonian translocation can experience multiple miscarriages because of the way the chromosomes segregate, leading to loss of a chromosome or a trisomy which is incompatible with life
Describe the link between Robertsonian translocation and trisomy 21
However, it becomes a problem in the context of forming gametes, because although there’s the correct amount of genetic material, the chromosomes can’t segregate properly.
If you’re lucky, the gamete will contain the normal chromosomes, or the robertsonian chromosome
If you’re unlucky then the gamete will contain one of these combinations.
Most of them will be lethal
But upon fertilisation with a normal gamete, this cell will have 2 copies of chromosome 14, which is fine, but 3 copies of chromosome 21 – and will therefore be a Down’s baby.
This will be a “normal” Down’s baby in that the phenotype will be similar to a Down’s which is the product of non-disjunction.
Approximately 4% of Down’s patients are because of Robertsonian translocations and 95% are due to non-disjunction.
What are the outcomes of translocations?
Very difficult to predict
Only have approximate probability of producing possible gametes
Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
Some unbalanced outcomes may lead to miscarriage later on and present clinically
Some may result in live-born baby with various problems
Describe other structural changes of chromosomes
There are some other types of structural changes which are summarised here
The first two are deletions, either from the end of the chromosome or from within a chromosome
If the end of the chromosome is lost then the only way the chromosome can be made stable is if a new telomere is added; without the telomere the cell will die
There are many examples of disorders caused by loss of chromosomal regions and I’ve listed a few here; you’ll learn more about those next year.
Inversions and duplications are literally as they’re described:
An inversion is where there are two breakpoints within the same chromosome and when these are repaired the middle section is “upside down”
A duplication is where you get a region of the chromosome repeated – you’ll probably be familiar with this in terms of the globin gene family
A ring chromosome is where you get two breaks in the same chromosome and that non-homologous end joining mechanism joins the two ends of the large chunk together, resulting in a ring.
Describe deletions
1:7000 live births
Deletion may be terminal or interstitial
Causes a region of monosomy
Haploinsufficiency of some genes
Monosomic region has phenotypic consequences
Phenotype is specific for size and place on deletion
Gross deletions seen on metaphase spread on G-banded karyotype
What are microdeletions and microduplications
Many patients had no abnormality visible on metaphase spread
High resolution banding, FISH and now CGH showed ‘micro’ deletions
Only a few genes may be lost or gained – contiguous gene syndrome
What is array CGH
Step 1-3:
Patient and control DNA are labelled with fluorescent dues and applied to the microarray.
Step 4: Patient and control DNA compete to attach or hybridize to the microarray.
Step 5: The microarray scanner measures the fluorescent signals.
Step 6: Computer soft eate analyses data and generates a plot.
These microarrays are created by the deposit and immobilization of small amounts of DNA (known as probes) on a solid support, such as a glass slide, in an ordered fashion. Probes vary in size from oligonucleotides manufactured to represent areas of interest (25–85 base pairs) to genomic clones such as bacterial artificial chromosomes (80,000–200,000 base pairs). Because probes are several orders of magnitude smaller than metaphase chromosomes, the theoretical resolution of aCGH is proportionally higher than that of traditional CGH. The level of resolution is determined by considering both probe size and the genomic distance between DNA probes. For example, a microarray with probes selected from regions across the genome that are 1 Mb apart will be unable to detect copy number changes of the intervening sequence.
What does congenital mean?
Present at birth
What does inborn mean?
Transmitted through the gametes.
What did Archibold E Garrod propose?
Garrod proposed that: Alkaptonuria Cytinuria Albinism Pentosuria Congenital (present at birth) Inborn (transmitted through the gametes) Had the discontinuous distribution of a Mendelian trait
What is alkaptonuria
AR Urine turns black on standing (and alkalinisation) Black ochrontic pigmentation of cartilage & collagenous tissue Arthritis (Homogentisic acid oxidase def.)
What is cystinuria
AR
1:7,000
Defective transport of cystine and dibasic aa’s through epithelial cells of renal tubule and intestinal tract
Cystine has low solubility -formation of calculi in renal tract
COLA or COAL
Mutations of SLC3A1 aa transporter gene (Chr 2p) & SLC7A9 (Chr 19)
What is albinism
Tyrosinase negative
Type 1a - complete lack of enzyme activity due to production of inactive tyrosinase
Type 1b - reduced activity of tyrosinase
Tyrosinase postive
Type II
AR, biosynthesis of melanin reduced in skin hair and eyes
most individuals do acquire a small amount of pigment with age
What is pentosuria
Excrete 1-4g pentose sugar L-xylulose daily (reducing sugar)
Benign
Almost exclusively Ashkenazi Jews of Polish-Russian extraction (1:2,500 births)
Describe the one gene-one enzyme concept
Beadle and Tatum 1945 (Nobel prize 1958)
all biochemical processes in all organisms are under genetic control
These biochemical processes are resolvable into a series of stepwise reactions
Each biochemical reaction is under the ultimate control of a different single gene
Mutation of a single gene results in an alteration in the ability of the cell to carry out a single primary chemical reaction
What is the molecular disease concept
Pauling et al 1949, Ingram 1956
Work on haemoglobin in sickle cell disease
Direct evidence that human gene mutations actually produce an alteration in the primary structure of proteins
Inborn errors of metabolism are caused by mutations in genes which then produce abnormal proteins whose functional activities are altered
What are the different mechanisms/types of inheritance
Autosomal recessive
Both parents carry a mutation affecting the same gene
1 in 4 risk each pregnancy
Consanguinity increases risk of autosomal recessive conditions
Examples: Cystic fibrosis, sickle cell disease
Autosomal dominant
Rare in IEMs
Examples: Huntingdon disease, Marfan’s, Familial hypercholesterolaemia
X-linked
Characterised by carrier females passing on condition to their affected sons
No male to male transmission
Female carriers may manifest condition –Lyonisation (random inactivation of one of the X chromosomes)
X-linked dominant : fragile X
X-linked recessive: haemophilia, Fabry’s disease
Codominant
two different versions (alleles) of a gene are expressed, and each version makes a slightly different protein. Both alleles influence the genetic trait or determine the characteristics of the genetic condition.
Example: ABO Blood group, α1AT
Mitochondrial
Mitochondrial DNA
Inherited exclusively from mother
only the egg contributes mitochondria to the developing embryo
only females can pass on mitochondrial mutations to their children
Fathers do not pass these disorders to their daughters or sons
Affects both male and female offspring Distribution of affected mitochondria determine presentation
High energy-requiring organs more frequently affected
Current debate on three parent babies
What are inborn errors of metabolism
Inborn errors of metabolism or inherited metabolic disease (IMD)
Individually rare (1:10,000 - 1:500,000)
Collectively present sizeable problem but cumulatively frequent and account for approx 42% of deaths within the first year of life
They make a significant contribution to the 1% of children of school age with physical handicap and the 0.3% with severe learning difficulties
Screening programmes
Important to recognise in sick neonate
What is the criteria for screening
-Wilson and Jungner
condition should be an important health problem
natural history of the condition should be understood
there should be a recognisable latent or early symptomatic stage
there should be a test that is easy to perform and interpret, acceptable, accurate, reliable, sensitive and specific
there should be an accepted treatment recognised for the disease
treatment should be more effective if started early
there should be a policy on who should be treated
diagnosis and treatment should be cost-effective
case-finding should be a continuous process
What is the WHO criteria for a good screening test
condition screened for should be an important one
there should be an acceptable treatment for patients with the disease
facilities for diagnosis and treatment should be available
there should be a recognised latent or early symptomatic stage
there should be a suitable test or examination which has few false positives -specificity - and few false negatives - sensitivity
test or examination should be acceptable to the population
cost, including diagnosis and subsequent treatment, should be economically balanced in relation to expenditure on medical care as a whole
Describe newborn blood spot screening properties
Initial National programme PKU Congenital hypothyroidism Extended to include Cystic fibrosis MCADD Haemoglobinopathies From 2015, the screening in England expanded to include four additional conditions Maple syrup urine disease (MSUD) Homocystinuria (pyridoxine unresponsive) (HCU) Isovaleric acidaemia (IVA) Glutaric aciduria type 1 (GA1)
Describe taking blood spots for screening
Samples should be taken on day 5 (day of birth is day 0)
All four circles on card need to be completely filled with a single drop of blood which soaks through to the back of the Guthrie card
What is the presentation of IEM
Neonate to adult Neonatal presentation often acute Often caused by defects in energy metabolism Maple syrup urine disease Tyrosinaemia OTC (urea cycle defect) Adult Wilson’s Haemochromatosis
Describe the presentation of neonates with IEM
Most are born at term with normal birthweight and no abnormal features
Symptoms frequently in the first week of life when starting full milk feeds
clues for IEMs
Consanguinity
FH of similar illness in sibs or unexplained deaths
Infant who was well at birth but starts to deteriorate for no obvious reason.
Classic presentation includes:
Full term pregnancy
Symtoms - can be very non-specific
Poor feeding, Lethargy, Vomiting, Hypotonia, Fits
or Specific
Abnormal smell (sweet, musty, cabbage-like)
Cataracts
Hyperventilation 2 to metabolic acidosis
Hyponatraemia and ambiguous genitalia
Neurological dysfunction with respiratory alkalosis
Biochemical abnormalities Hypoglycaemia Hyperammonaemia Unexplained metabolic acidosis / ketoacidosis Lactic acidosis Clinical Cognitive decline Epileptic encephalopathy Floppy baby Exercise intolerant Cardiomyopathy Dysmorphic features SUDI Fetal hydrops
Types of laboratory investigations
Routine laboratory investigations
- Blood gas analysis
- Blood glucose
- Plasma ammonia
Specialist investigations
- Plasma amino acids
- Urinary organic acids + orotic acid
- Blood acyl carnitines
- Blood lactate and pyruvate
- Urinary glycosaminoglycans
- Plasma very long chain fatty acids
Enzymology
- Red cell galactose-1-phosphate uridyl transferase
- Lysosomal enzyme screening
Biopsy (muscle, liver)
Fibroblast studies
Complementation studies
Mutation analysis – whole genome sequencing
What are the possible metabolic causes for acute liver disease in neonates?
Classical galactosaemia
Hereditary fructose intolerance
An organic acidaemia
Tyrosinaemia type 1
What is tyrosinaemia type 1?
Blockage in metabolism of tyrosine by reduced fumaryloacetate enzyme.
Get overforming of succinylacetoacetate and then succinylacetate.This blocks alanine from converting to Porphobilinogen.
Define:
DNA
GENES
THE HUMAN GENOME
DNA
A molecule which contains the human genetic code
Genes
The instructions to tell the body how to grow, develop and function
Consist of sections of DNA which the cell translates into proteins
~ 20,000 genes in the human genome
2 copies of most genes – one on each chromosome
A section of the genetic code which can be translated into a protein.
Three letters code for one amino acid, and a string of amino acids make up a protein
The human genome
The entire length of DNA contained in human cells
3 billion bases
how has genetic technologies influenced us?
Over time, the technologies we have to interrogate our DNA have been getting cheaper and faster
As our understanding has improved, we have discovered more disease genes
Before, we could only see the genome using a microscope to see chromosomes
Now we can see sections of chromosomes (FISH, microarray) and sequence whole genomes in 1-2 days. It took billions to pounds to sequence the genome for the whole genome project.
How has genetic variation effected us?
External factors like smoking, exposure to UV
Causes a permanent change to the DNA, known as a mutation.
Makes us unique
“polymorphisms”
Is the basis for evolution
Is the basis for disease
Caused by intrinsic errors in DNA replication and repair
Caused by external factors
Describe cancer
Normal cells divide, replicating their DNA before division
DNA replication is complicated and can result in errors in a gene/s (i.e. a somatic mutation)
Normal cells die when an error cannot be repaired
Cancer results when mutations accumulate, cell does not die and cell growth is uncontrolled.
Somatic mutation – just in that one cell but not in every cell in the body.
What are the current cancer statistics
Cancer is a common disease in humans
There is a 1 in 2 lifetime risk of developing cancer
Most cancers are caused by a combination of genetic, environmental and lifestyle factors – multifactorial/sporadic
Only ~5-10% of cancers are due to the inheritance of a single cancer susceptibility gene.
Describe the difference between germ line and somatic mutations?
Multifactorial/sporadic cancers
Somatic mutations Occurs after division of the fertilised egg Only present in a subset of cells Not inherited from a parent Occasionally passed to offspring Occur in non germline tissues Cannot be inherited. Mutation in tumor only (eg breast)
Germ line mutations: Present in egg or sperm Can be inherited Cause cancer family syndrome. If mutation in egg or sperm, all cells affected in offspsring.
Hereditary cancers:Hereditary cancers Germline mutations +/- somatic mutations Present in the fertilised egg Present in every cell in the body Can be inherited from a parent Can be passed to offspring
What are differences between sporadic and hereditary cancers
Sporadic cancers
No increased risk of other cancers
Usually small increased risk to relatives
No genetic testing indicated
Normal clinical management for affected individuals
Hereditary cancers
High risks of recurrence/other associated cancers
High cancer risks in relatives
We can offer testing to at risk individuals
We can offer screening and preventative management to gene carriers
May alter treatment of affected individuals
Knudson’s ‘Two hit hypothesis’
RB is a rare tumour of the eye
RB gene is a tumour suppressor gene where both copies have to be faulty to develop the disease
Hereditary RB occurs ay younger ages and often affects both eyes compared to nonhereditary where both mutations are somatic
What is penetrance
NOT every person with a germline mutation develops the disease
Known as reduced penetrance
We can give risks of developing disease for a given genotype
Based on family/population studies
Unknown modifying factors
Describe the estimated proportion of hereditary cancers
Breast cancer 5-10% Ovarian 10% Colon 5-10% Melanoma 10% Medullary thyroid 25% Retinoblastoma 40% Prostate 5-10% Pancreatic 10% Different genetic component to different cancers – ¼ medullary thyroid cancers are due to germline mutations
Cancer susceptibility genes
Different classes of genes are targeted in cancer, which function in normal cell regulation
Growth promoting proto-oncogenes
E.g. RET in MEN2
Growth inhibiting tumour suppressor genes
E.g. RB1 in retinoblastoma
Genes involved in DNA damage repair
E.g. BRCA1 and BRCA2 genes in breast/ovarian cancer
Other mechanisms of oncogenesis
Epigenetic mechanisms of oncogenesis
Chromosomal aberrations
Normal cells are tightly regulated to prevent cancer
How do we distinguish hereditary cancers from sporadic cancers
Taking a family history 3 generation family history
Ask about consanguinity
Ethnic background
Ashkenazi Jewish, other founder populations
Types and ages of all cancers
NB. Some individuals with a hereditary predisposition to cancer do not have a family history of cancer
In genetics, clues to an underlying genetic susceptibility come from the family history
What are the decisions to make from family history
Is genetic testing indicated?
other investigations required first?
Confirmation of cancer diagnoses
Testing of tumour samples (e.g. IHC)
Is increased screening indicated?
For affected individual
For unaffected relatives
How do we undertake genetic testing
Implications for individual Recurrence risks Risks of other cancers Implications for relatives How to share information Concerns about children Predictive testing Insurance implications Current moratorium for predictive testing Family planning options (e.g. prenatal, PGD)
BRACA1 and BRACA2 genes
BRCA1 mutations - 0.11% population
BRCA2 mutations - 0.12% population
Function in repair of double stranded DNA breaks (homologous recombination)
Responsible for ~ 16% familial breast cancers ~ 5% breast cancer ~ 10% ovarian cancer Also prostate, pancreatic, fallopian tube and peritoneal cancers
-graph to show increase risk in breast and ovarian cancer on phone in favourites.
Describe the screening for BRCA carriers
Breast Screening
30-50y annual MRI screening
30-50y annual mammograms
>50 annual mammogram
Ovarian Screening
Unproven efficacy
Not currently recommended
What are the surgical options for breast cancer?
Risk reducing mastectomy Most effective way of reducing risk - <5% over lifetime Avoids need for cancer treatment Can help women with anxiety Breast reconstruction available
What are the surgical options for ovarian cancer?
Risk reducing bilateral salpingo-oophorectomy
Offered at age 40 or after completed family
Can give HRT under specialist guidance
Only proven way to reduce ovarian cancer risk
What are other clinical managements of cancer?
Altered clinical management:PARP inhibitors
Sensitivity to platinum chemotherapies
Describe hereditary non polyposis colorectal cancer syndrome (HNPCC)
Lynch syndrome
Germline mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2)
Hereditary predisposition to:
colorectal cancer
ovarian cancer
endometrial cancer
Plus gastric, pancreatic, hepatobiliary tract, urothelial and small intestine cancers
Screening for HNPCC
Colonoscopy every 18-24 months, from age 25-30
Discuss endometrial screening from age 35 (but not proven to be effective)
Discuss option of risk-reducing TAH/BSO from early 40’s
How do we test for Lynch syndrom
Tumour testing on colorectal and endometrial cancers (IHC/MSI)
Gene panel testing for MLH1/MSH2/MSH6/PMS2
Describe prophylactic management
Aspirin reduces cancer risk in Lynch syndrome patients by ~50%
CAPP2 research study showed aspirin reduced risk of colorectal cancer
CAPP3 research study
600mg, 300mg, 100mg aspirin daily
Examples of other hereditary cancer syndromes
Li-Fraumeni Syndrome (TP53 gene)
Adrenocortical, sarcomas, childhood, breast
Highly penetrant (70-90% chance of cancer)
Renal cancer syndromes (VHL, FLCN, FH, MET)
Pheochromocytomas/Paragangliomas (SDHB/C/D)
Multiple Endocrine Neoplasia Type 1 (MEN1)
Multiple Endocrine Neoplasia Type 2 (RET)
Retinoblastoma (RB1)
Neurofibromatosis Type 1 (NF1)
Neurofibromatosis Type 2 (NF2)
Tuberous Sclerosis (TSC1/TSC2)Familial Adenomatous Polyposis (AFP)
MYH-associated Polyposis (MUTYH)
Peutz-Jeghers syndrome (STK11)
Cowden Syndrome (PTEN)
Thyroid and breast cancers, macrocephaly
Diffuse stomach cancer and lobular breast cancer (CDH1)
Familial melanoma (p16)
