1. Mrs Jones’ First Consultation

MCD Genetics
Alexandra Burke-Smith
1. Mrs Jones’ First Consultation
Dr Andrew Walley (a.walley@imperial.ac.uk)








She is pregnant
She has heard that 1 in 50 babies born have a congenital malformation
Her uncle has haemophilia
Her husband’s first cousin has a child with Cystic Fibrosis
She is 35 (relatively old for pregnancy)
This is her first pregnancy
She is 7 weeks pregnant
Her mother had 4 miscarriages and 4 normal children (family history needs to be taken into account)
1. Congenital Abnormalities



Congenital abnormalities are apparent at birth in 1 in 50 of all newborn infants
20-25% of all deaths during perinatal period and childhood up to the age of 10 years
Genetic factors contribute to about 40% of all congenital abnormalities
1. Malformation – primary structural defect e.g. atrial septal defects, cleft lip. This usually involves a single
organ showing multifactorial inheritance (i.e. not just genetic)
2. Disruption – secondary abnormal structure of an organ or tissue e.g. amniotic band causing digital
amputation. Caused by ischaemia (inadequate flow of blood to a particular body part), infection, and
trauma. Not genetic, but genetic factors can predispose.
3. Deformation – abnormal mechanical force distorting a naturally formed structure e.g. club foot, hip
dislocation. Occurs late in pregnancy and has a good prognosis as the organ is normal in structure, just
physically malformed
4. Syndrome – consistent pattern of abnormalities with a specific underlying cause, e.g. Down syndrome.
Collection of abnormalities, usually genetic e.g. Chromosomal abnormalities
5. Sequence – multiple abnormalities initiated by primary factor e.g. reduced amniotic fluid leads to Potter
sequence. Could have genetic component as initial factor, i.e. not due to a single genetic initially, e.g.
Oligohydramnios – reduced volume of amniotic fluid due to failure to produce urine, which is classically due
to bilateral renal agenesis (Potter, 1946)
6. Dysplasia –abnormal organisation of cells into tissue e.g an abnormal development of epithelium, bone or
other tissues such as thanatophoric dysplasia (severe skeletal disorder characterized by extremely short
limbs and folds of extra (redundant) skin on the arms and legs), as well as a large head and a small thorax. Is
caused by single gene defect in the FGFR3 gene, and carries a high recurrence for siblings and offspring of
the affected person. This gene provides instructions for making a protein that is involved in the development
and maintenance of bone and brain tissue. Mutations in this gene cause the FGFR3 protein to be overly
active, which leads to the severe disturbances in bone growth that are characteristic of thanatophoric
dysplasia.
7. Association –non-random occurrence of abnormalities not explained by syndrome. Cause is typically
unknown. e.g. VATER association:
Vertebral Anal Tracheal Esophageal Renal.
It is a non-random association of birth defects. The reason it is called an association, rather than a syndrome
is that while all of the birth defects are linked, it is still unknown which genes or sets of genes cause these
birth defects to occur. Classification of an association is not mutually exclusive (i.e. can get one as a result of
another) e.g a primary malformation of kidneys can lead to the same sequence of events as Potters
syndrome – risk estimates of inheriting the disorder is therefore a problem.
1
MCD Genetics
Alexandra Burke-Smith
8. Dysmorphism – an unusual or abnormal physical feature (sometimes as part of a genetic syndrome) e.g.
hypertelorism (abnormality which results in an increased distance between two organs e.g. eyes)
2. Chromosomes and Genetics



DNA contains genes which are packaged as chromosomes, all of which make up the human genome.
Chromosome numbers: the diploid number you inherit makes up the entire genome. In humans this is 22
autosomes and one sex chromosome from each parent (23 pairs)
DNA Packaging problem: 2 metres of DNA in each cell of the body, therefore extreme packaging must occur
to fit into a chromosome.
The chromosome
Long thread-like structure composed of DNA and associated proteins that carries the genetic information of an
organism. There are 3 types:



Metacentric: two equally long arms
Submetacentric: one set of “short arms”
Acrocentric: one set of chromatids (arms) virtually non existant- but rather seen as “satellites”
Banding Nomenclature: Bands are labelled according to the chromosome number, short (p) or long (q) arm and
numbered out from the centromere . This is used to identify different chromosomes.
Imaging: FISH (Fluorescent in-situ hybridisation) is a way of probing chromosomes, highlighting specific areas on the
chromosome e.g. the centromere.
The human karyotype: a display of the full set of chromosomes of a cell arranged with respect to size, shape and
number.
3. Chromosome Abnormalities
Chromosome abnormalities are present in: 60% of early spontaneous miscarriages, 4-5% of still births, 7.5% of all
conceptions, and 0.6% of live births
There are three types:
Numerical –
aneuploidy, loss or gain (change in total number)
Structural –
translocations, deletions, insertions, inversions, rings
Mosaicism – different cell lines
Autosomal Aneuploidy
•
•
•
Monosomy - loss of a single chromosome is almost always lethal
Trisomy - gain of one chromosome can be tolerated
Tetrasomy - gain of two chromosomes can be tolerated
The loss of a chromosome gives a reduction of 50% of all fully expressed gene products, whereas the gain of one
chromosome gives an increase of 33% of all fully expressed gene products. Therefore trisomers are more common
than monosomers as it results in a smaller change in the expressed gene.
Translocations, i.e. partial aneuploidy
• Balanced: the “swap of areas” on the homologous chromosomes during meiosis. The chromosomes are still
a normal length therefore this is unlikely to have a significant effect.
• Unbalanced: the swap results in the chromosomes not being of normal length, and the total DNA on each
chromosome is not equal, therefore they are more likely to cause disease
Trisomy 21: Down syndrome
• Overall incidence at birth is approx 1 in 650 to 1 in 700
• Strong association between incidence and advancing maternal age, i.e. >40 poses significant risk
2
MCD Genetics
•
Alexandra Burke-Smith
Clinical features:
Newborn period - sleepy, excess nuchal skin
Craniofacial - macroglossia, small ears, epicanthic folds, upward sloping palpebral fissures, Brushfield spots
Limbs – single palmar crease, wide gap between first and second toes
Cardiac - A and V septal defects
Other - short stature, duodenal atresia
NB: Nuchal is back of neck
Macroglossia is the medical term for unusual enlargement (hypertrophy) of the tongue.
Palpebral fissue is the gap between upper and lower eyelids
Brushfield spots are characteristic white spots in iris
Atresia is a condition in which a body orifice or passage in the body is abnormally closed or absent
•
•
•
•
•
•
IQ scores 25-75
happy and affectionate
Relatively advanced social skills
Adult height around 150 cm
Relatively normal life expectancy but Cardiac anomaly causes early death in 20%
Increased risks of leukaemia and Alzheimer’s
•
95% of all Down cases are caused by non-disjunction (not splitting properly)
during meiosis
4% of all cases are caused by translocations; the breakage of acrocentric
chromosomes and fusion of their long arms
1% of all cases are caused by mosaicism; occurs after the zygote is formed, and depending on when it occurs,
you will be able to determine the proportion of affected cells in the body.
•
•
Monosomy X: Turner’s syndrome
• 1 in 3000 live female births
• Generalised oedema and swelling in neck region can be detected in 2nd trimester
• Can look normal at birth or have puffy extremities and intra-uterine oedema
• Low posterior hairline, short 4th metacarpals, webbed neck, aorta defect in 15% of cases
• Normal intelligence
• In adults:
• short stature - 145 cm without Growth Hormone treatment due to loss of SHOX gene
• ovarian failure - primary amenorrhoea and infertility
• Treatment -oestrogen replacement for secondary sexual characteristics and prevention of osteoporosis
• 80% due to loss of X or Y chromosome in paternal meiosis
• Also ring chromosome, single arm deletion, mosaicism in X chromosome
Ring Chromosome: Breaks occur on the ends of the two arms of a chromosome and the sticky ends are then joined
and the fragments are lost. These are often unstable at mitosis and so mosaicism is frequent. Some cells have the
ring and the rest are monosomic.
Sex Chromosome Aneuploidy
• Female has two X chromosomes, but other one is required. The Y chromosome is short and carries very few
genes, carries SRY gene- determines maleness
•
Polysomy X in females: 47,XXX karyotype
10-20 point decrease in IQ
No physical abnormalities
95% have extra maternal X arising in meiosis I
Normal fertility
48,XXXX and 49,XXXXX karyotypes show mental retardation
Not as devastating due to X-inactivation
3
MCD Genetics
•
Alexandra Burke-Smith
Polysomy X in males: Klinefelter’s syndrome (47,XXY)
1 in 1000 male live births
o clumsiness, verbal learning disability 10-20 pts
o taller than average (long lower limbs)
o 30% - moderately severe gynaecomastia
o all infertile
o increased risk of leg ulcers, osteoporosis and breast carcinoma in adult life
X chromosome from either Male or Female
48,XXXY and 49,XXXXY are rare
4. Sex Determination
•
•
Chromosomal Sex and Gender: it is possible to be chromosomally one gender and phenotypically the
opposite
SRY gene activated at 6 weeks post-conception signalling the development of the testes
XX males: Translocation of the SRY male determining gene from Y chromosome to an X chromosome. Phenotypically
male, testes develop, but sterile because some genes on Y chromosome needed for spermatogenesis.
XY females: Mutations or deletions of SRY gene leads to phenotypically female who is infertile
5. Genomic Disorders
Recurrent Microdeletion Disorder: due to very small (not visible on the karyotype) deletions in the DNA sequence
•
•
Di George Syndrome: most common microdeletion disorder. Approx 1/4000 live births. Variable symptoms
such as Congenital Heart Disease,Palatal abnormalities
Cri du Chat Syndrome: Severe psychomotor and mental retardation. Characteristic Facies. Characteristic catlike cry in newborns. Rare – Approx 1 in every 50000 live births.
Mrs Jones
 She has elevated risks of miscarriage and congenital abnormalities because of her age and family history
 We need to further investigate the fact that she has mentioned haemophilia and cystic fibrosis within her
family
4
MCD Genetics
Alexandra Burke-Smith
2. Mrs Jones (2)- risk of transmission of
genetic disease
Dr Andrew Walley (a.walley@imperial.ac.uk)
1. Genetic Disease
Monogenic disorders
 Are familial, i.e. occur as a direct consequence of a single gene being defective and are passed on from one
generation to the other
 They have a specific mode of inheritance e.g. Mendelian
 They can be common and rare
Eg. Huntington disease, Cystic fibrosis, Haemophilia
Complex disorders
 likely associated with the effects of multiple genes in combination with lifestyle and environmental factors.
 Although complex disorders often cluster in families, they do not have a clear-cut pattern of inheritance.
 Can also be sporadic
 Influenced by environmental factors e.g. diet, lifestyle etc
 Cause of many common disorders
Eg. Type 2 diabetes
Obesity
Parkinson’s disease
Mendelian Inheritance
The process whereby individuals inherit and transmit to their offspring one out of the two alleles present in
homologous chromosomes.
Alleles





Alternate forms of gene or DNA sequence at the same chromosome location (locus)
Homologous chromosomes are a matching (but non-identical) pair, one inherited from each parent
Homologous chromosomes can have different alleles
There can also be multiple alleles present at the same locus
Different alleles may be described as mutations or polymorphisms
Mutation: any heritable change in the DNA sequence. This is a change in the genotype which has a definite effect on
the phenotype, e.g. causing disease
Polymorphism: The occurrence of a chromosome or genetic characteristic in more than one form, which results in the
coexistence of different phenotypes within a population, e.g. different hair colours. Polymorphisms may contribute to
complex diseases
Types of Mutation
 Missense: a point mutation (change in a single base pair) which then codes for a different amino acid. This
doesn’t mean it will definitely affect the protein function, for example if the change did not affect the active
site.
 Nonsense: a point mutation results in the formation of a stop codon, which leads to premature termination
of the polypeptide chain, which can have a significant effect on the function of the protein.
 Insertion: of a nucleotide can create a frameshift, as the genetic code is read in triplets. This can result in a
completely different protein being coded for
 Deletion: can also create a frameshift, but if 3bp are deleted this may have no effect.
5
MCD Genetics
Alexandra Burke-Smith
2. Pedigree Diagrams
Why draw them?
 To identify genetic disease
running in family (complex and
monogenic)
 To identify inheritance patterns
 To aid diagnosis
 To assist in management of
conditions
 To identify relatives at risk of
disease
1) Build up the tree from the
‘bottom’ starting with affected
child and siblings
2) Record names, dates of birth
3) Choose one parent. Ask about
sibs and their children, then parents
4) Record names, dates of birth and maiden names (could be used to identify common ancestors which can
help with inheritance pattern)
5) Ask for miscarriages, stillbirths or deaths in each partnership
6) Ask about children through other partnerships
3. Mendelian Inheritance Patterns
Autosomal Dominant
 At least one affected parent- doesn’t skip generations
 Transmitted by Male or Female
 Vertical transmission
 Males or Females affected
 Each child has a 50% chance of being affected
E.g. Huntington Disease
• Motor, cognitive, and psychiatric dysfunction: ‘hyperkinesia’ (excessive uncontrollable movements)
• Affects dopamine signalling in the basal ganglia
• Mean age of onset is 35 to 44 years, therefore it is often passed on as parents have children before the onset
• Median survival time is 15 to 18 years after onset
• Treatment can ease symptoms, but no cure
HTT gene on chromosome 4 encodes a protein called huntingtin
 HD patients inherit one copy of a mutated form of the huntingtin gene
 altered gene encodes a toxic form of the protein that form ‘clumps’
 cell death in basal ganglia of the brain, leading to symptoms
Dominant Anticipation
Anticipation is the increase in severity and/or earlier onset of symptoms in each generation
- Each generation gets it younger
- Huntington Disease is caused by an unstable CAG triplet repeat (coding for glutamine): the number of
repeats may expand with each generation as DNA polymerase loses its place in the sequence.
Autosomal Recessive
•
•
•
•
No affected parent
Transmitted by M or F
Usually no family history
M or F affected
6
MCD Genetics
Alexandra Burke-Smith
• 25% of children affected
• 50% inherit one copy of defective gene; i.e. they are carriers
• Requires two affected genes
• Requires genetic testing
• Tends to be more frequent where an element of consanguinity is involved
E.g. Cystic Fibrosis
• A chronic, life-threatening condition
• Thick mucus in lungs causes breathing problems and repeated infections
• Blockages in pancreas affect digestive enzymes
• Treatment consists of daily enzymes and physiotherapy
• In the UK, 1 person in 22 is a CF carrier (no symptoms)
• Most common mutation is the deletion of phenyl alanine (delta F508) which affects folding of CFTR protein
and prevents it from moving to its correct place (the cell membrane)
• The CFTR gene on chromosome 7 encodes a protein called the CF transmembrane conductance regulator
 CF patients inherit two copies of a mutated form of the CFTR gene
 Absence of any working CFTR protein affects chloride ion channel function in ‘wet’ epithelial cells
 Disruption of salt /water regulation causes thick mucus and leads to symptoms
• CF testing now part of UK newborn screening programme
•
•
•
•
Congenital absence of the vas deferens (CAVD) is a condition in which the vasa deferentia fail to form
properly, therefore sperm are made but are not transported to the epidymis.
Causes infertility (azoospermia)
Affects around 1 in 2500 men
Most cases of CAVD are caused by mutations in the CFTR gene
X-linked disorders
•
•
•
•
•
•
•
No affected parents
M affected
Transmitted by carrier F
50% sons affected
50% daughters carriers
Mutations on X chromosome
Majority are recessive, and as there is no homologous section on the Y chromosome therefore
expressed
E.g. Haemophilia
• A blood-clotting disorder
• Affected people bruise easily, and bleed for longer
• Two main types, A and B, which together affect about 6500 people in the UK
• Can be successfully treated with injections of clotting factor
• The F8 gene on the X chromosome a protein called coagulation factor VIII
 Boys with Haemophilia A inherit patients inherit one copy of a mutated form of the F8 gene
Lack of functioning Factor VIII causes symptoms of disorder
• X-linked because the genes involved are located on the X-chromosome, recessive because carrier females
are unaffected (they have a working F8 or F9 gene on their other X-chromosome
• Haemophilia B is caused by mutations in the F9 gene, also on the X chromosome
• F9 gene codes for coagulation factor IX
• Symptoms are identical to those of Haemophilia A
• Haemophilia B is much rarer than Haemophilia A
Genetic Heterogeneity
•
•
•
•
Same gene, different mutations, different diseases - eg. cystic fibrosis and CAVD are both caused by
mutations in the CFTR gene
Same disease, different genes – eg. Haemophilia A (mutations in F8 gene) and Haemophilia B (mutations in
F9 gene)
Same disease, different genes, different inheritance patterns – eg. different forms of epidermolysis bullosa
can be autosomal dominant or autosomal recessive
This adds complexity
7
MCD Genetics
Alexandra Burke-Smith
Complexity of Inheritance Patterns
•
•
•
•
Penetrance – frequency with which symptoms are present in an individual who inherits a disease-causing
mutation, i.e. frequency of phenotype associated with a particular genotype
Variable expressivity – degree of severity in an individual who inherits a disease-causing mutation
Phenocopy - disease with the same phenotype as a genetic disease, but non-genetic, e.g. autoimmune form
of Haemophilia
Epistasis – interaction between disease gene mutations and other modifier genes can affect phenotype
4. Mechanisms of genetic disease
•
•
•
Dominant conditions: usually caused by gene mutations that results in a toxic protein (eg. HD) – ie. effects
of mutated gene ‘mask’ normal copy
Recessive conditions: Caused by absence of working protein (eg. CF, haemophilia) – ie. effects of mutated
gene only seen when normal copy absent
Co-dominant conditions: Effects of both mutated and normal genes apparent in people with both, e.g. sickle
cell trait, ABO blood grouping
Implications for Therapy
•
•
Dominant conditions – need to counter effects of, or neutralise toxic protein, or ‘switch off’ mutant gene,
therefore harder to treat
Recessive conditions – need to restore activity of missing protein, by replacing genes, protein or affected
tissues, e.g. use of replacement clotting factors in Haemophilia
Mrs Jones
Haemophilia
•
Her uncle has haemophilia BUT its her paternal uncle. Therefore the chance of a son with haemophilia
requires a new mutation
Cystic Fibrosis
•
•
•
•
•
•
Her husband’s first cousin has a child with CF therefore Mrs Jones’ husband has a 1:8 chance of being a CF
carrier. (1/2 chance from each parent, and ½ chance of being passed onto next generation therefore ½ x ½ x
½ = 1/8)
Mrs Jones has a 1:22 chance of being a CF carrier (UK population chance)
If both are carriers there is a 1:4 the child is affected
Therefore the overall chance of affected foetus = 1/704 = 1/8 x ½ x 1/22
The overall chance that foetus is affected by CF is based on chance that both parents are carriers – without
genetic testing we cannot tell if the husband is a carrier or not – multiplied by the individual risk to each
child
This proves the need for a detailed family history
8
MCD Genetics
Alexandra Burke-Smith
3. More Stories from the Genetic Clinic
Dr Andrew Walley (a.walley@imperial.ac.uk)
1. Imprinting Disorders
Genetic imprinting refers to a situation where genes are expressed differently according to whether they are inherited
on the chromosome that came from the mother or that from the father





There are cases of 46,XX where genome is only from one parent
- Maternal – Ovarian Teratoma
- Paternal – Hydatidiform Mole
The genome carries an imprint of its parental
origin
Imprinting is a reversible epigenetic effect, i.e. it
is not a result of a change in the primary
sequence of DNA, but rather through DNA
methylation of cytosines.
The imprinted domain is the place where DNA
Methylation occurs
Uniparental Isodisomy: Non-Disjunction in
Meiosis II (i.e. failure of chromosomes to
separate). Fertilisation of normal monosomic
gamete then occurs, but there is a loss of
chromosome from parent contributing the single
chromosome.
Prader-Willi Syndrome vs Angelman Syndrome



Two distinct clinical syndromes
Same chromosomal region involved on Chr15
Result from loss of function of one of the two parental chromosomes
- Paternal = Prader-Willi
- Maternal = Angelman
9
MCD Genetics
Alexandra Burke-Smith
Prader- Willi (PWS)
Symptoms include
 Muscle hypotonia
 Hyperphagia (all-consuming appetite)
 Obesity/Type 2 Diabetes
 Mental retardation
 Short stature
 Small hands and feet
 Delayed/Incomplete Puberty
 Infertile
Prevalence
 1:10,000 to 1:25,000 for birth incidence
Management
 Hyperphagia managed by diet restriction
 Exercise to increase muscle mass and to combat
Hypertonia
 Growth Hormone treatment for short stature
 Hormone replacement at puberty
Diagnosis
 Methylation-specific PCR (polymerase chain
reaction)
Genetic Mechanism
Lack of a functional paternal copy of the PWS critical
region on 15q11-q13 chromsome
 ~70% result from deletion of the critical region on
the paternal chromosome
 ~25% result from inheritance of two maternal
copies by uniparental isodisomy
 ~5% are due to translocations, point mutations
Angelman
Symptoms include
 Severe developmental delay
 Poor or absent speech
 Gait ataxia (uncontrollable unsteady movements
that result from the brain’s failure to regulate the
body’s posture, strength and direction of
movements)
 “Happy demeanour”
 Microcephaly (abnormal smallness of the head)
 Seizures
Prevalence
 1:10, 000 (so similar prevalence to PWS, but more
severe)
Management
 Symptomatic treatment– anti-convulsant,
communication therapy
 Physiotherapy for gait ataxia
 Normal life span
Diagnosis
 Clinical Features and Molecular Diagnostics
Genetic Mechanism
Lack of a functional maternal copy of the PWS critical
region on 15q11-q13 chromsome
2. Mitochondrial Disorders
Mitochondrial Inheritance






Inheritance pattern quite distinctive
Initial mutation often in protein pumps present in the mitochondria (important in respiration etc)
Transmission is exclusively through females- sperm may contain mitochondria for movement, but they do
not inject their mitochondria into the ova.
Affects both males and females
Cells vary in their number of mitochondria depending on energy requirements; Numbers from 1 to>1000 per
cell
Disease can be very variable because of heteroplasmy- there are different numbers of mitochondria in
different cells, therefore only certain cells will be affected by the disease
Mitochondrial Genome







~16kb
37 genes
13 for respiratory chain complexes
22 for tRNA
2 for rRNA
2-10 copies per mitochondrion
Replicates its own DNA
10
MCD Genetics


Alexandra Burke-Smith
Most of the active genes/proteins are encoded by the nucleus
Disorders typically occur by point mutations
Mitochondrial Disorders
•
•
•
•
•
•
MELAS
LHON
MERRF – Myoclonic Epilepsy with
Ragged Red Fibres
DEAF – Non-syndromic hearing loss
NARP – Neuropathy, Ataxia and Retinitis Pigmentosa
MELAS
Mitochondrial myopathy, Encephalopathy, Lactic
Acidosis and Stroke
Progressive neurodegenerative disorder. Symptoms
include
 Muscle Weakness
 Episodic Seizures and headache
 Hemiparesis- paralysis of one side of the body
 Vomiting
 Dementia
Prevalence
 Estimated prevalence of 1:13 000
Management
 Symptomatic Treatment
Diagnosis
 Diagnosis by muscle biopsy
Genetics
 Single point mutations in several genes
 MTTL1 – tRNA translates codon as Phe instead of
Leu during mitochondrial protein synthesis
 MTND1, MTND5 – NADH dehydrogenase (Complex
1)
LHON
Leber’s Hereditary Optic Neuropathy
 For unknown reasons, this is much commoner in
males (suggests X-linked effect)
 Average age is mid-twenties to mid-thirties
 Age range is wide though (6-62yrs)
Symptoms include
 Bilateral, painless, loss of central vision and optic
atrophy
 Most will become blind
 Typically one eye will be affected first
Prevalence
 Estimated prevalence of 1:50 000
Management
 Symptomatic Treatment
Diagnosis
 Diagnosis is on the basis of opthalmological findings
and a blood test for mtDNA (mitochondrial DNA)
mutations
Genetics
 >90% of the mutations are in
o MTND1, MTND4, MTND5, MTND6 and MTCYB
o NADH dehydrogenase subunits 1,4,5 and 6
o Cytochrome B
11
MCD Genetics
Alexandra Burke-Smith
3. Inborn Errors of Metabolism




“one gene-one enzyme” concept
More than 200 diseases knownMostly autosomal recessive or Xlinked. A few are dominant (ratelimiting step or part of a multimeric
complex)
The defective proteins are mainly
enzymes found in the mitochondria
These are relatively easy to treat if
found early
4. UK Newborn Screening Programme
Introduced as early diagnosis improves
treatment. Screens newborns for :
 Phenylketonuria (PKU)
 Congenital Hypothyroidism
 Sickle Cell Disorders
 Cystic Fibrosis
 Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCAD Deficiency)
Phenylketonuria (PKU)






Occurs relatively quickly after birth
Severe mental retardation and convulsions
Blond hair/blue eyes; eczema
Phenylalanine hydroxylase deficiency, therefore
phenylalanine is unusually processed, i.e. normal
metabolism does not occur and compensatory
mechanisms occur.
Phenylalanine accumulates and is converted to
phenylpyruvic acid - excreted in urine
Tyrosine deficiency - reduced melanin and
accumulation of homogentisic acid
Thyroxine definciency- hormone involved with increase of the basal metabolic rate.
Alkaptonuria- accumulation of homogentisic acid causes dark brown discoloration of the skin and eyes, and
progressive damage to the joints, especially the spine.
Treatment
 Newborn screening for elevated levels of phenylalanine in blood
 Remove phenylalanine from diet
 Difficult diet to stick to. Best foods to eat are fruits and vegetables- difficult with children.
12
MCD Genetics


Alexandra Burke-Smith
Aspartame contains phenylalanine
Pregnant women need to go back on diet
MCAD Deficiency









Commonest disorder of fatty-acid oxidation
Episodic hypoketotic hypoglycaemia (no ketones,
blood sugar)
Commonly presents > 3 months
Frequency of 1:8000 to 1:15000 births
Can present as coma, metabolic acidosis,
encephalopathy
Sudden death can occur, with a 25% mortality rate
undiagnosed cases- major reason for screening
MCAD is Medium-Chain Acyl-CoA Dehydrogenase
MCAD gene is called ACADM
Treatment simple- maintains diet of readily usable
glucose energy. Maintenance of adequate calorie
intake to prevent switch to fatty acid oxidation, and
avoiding fasting.
low
in
13
MCD Genetics
Alexandra Burke-Smith
4. Cancer in families and Individuals
Dr Alistair Reid (a.reid@imperial.ac.uk)
1) Explain the terms somatic mutation and loss of heterozygosity and explain their implications for oncogenesis and
cancer progression
2) Discuss how mutations in BRCA1 and BRCA2 genes influence risk of breast and ovarian cancer
3) Outline how defects in DNA repair influence risk of colorectal cancer Explain the terms somatic mutation and loss
of heterozygosity and explain their implications for oncogenesis and cancer progression
4) Describe, using specific examples, how acquired genetic changes are used as disease markers
Overview
Cancer is driven by an accumulation of genetic or epigenetic changes that lead to altered levels of transcription
and/or aberrant gene transcripts. These activate signal transduction pathways that confer a selective advantage to
the cell, e.g. drug resistance. Cancer may be sporadic
or familial, and mutations may be infected or
acquired
Somatic mutations: Alterations in DNA that occur
after conception. Somatic mutations can occur in any
of the cells of the body except the germ cells (sperm
and egg) and therefore are not passed on to children.
These alterations can (but do not always) cause
cancer or other diseases.
Oncogenes and Tumour Suppressors
Normal functions of TS genes
 Regulating cell division
 DNA damage checkpoints (damage=no division)- After DNA damage, cell cycle checkpoints are activated.
Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to
divide. DNA damage checkpoints occur at the G1/S (Gap 1/Synthesis) and G2/M (Gap 2/ Mitosis) boundaries.
An intra-S checkpoint also exists
 Apoptosis: normal, benign type of programmed cell death in which a cell shrinks, fragments its DNA, and
alters its surface so as to activate the cell’s phagocytosis by macrophages
 DNA repair
 Mutations in the TS genes result in an inactivation or deletion of the gene, leading to unregulated cell
division
Normal oncogene functions
 An oncogene (ONC) is a gene that can cause cancer.
 It results from the mutation of a normal gene (proto-oncogene)
 An oncogene is capable of both growth and proliferation of malignant transformation of normal cells
 It produces proteins (e.g. Growth factors, Transcription factors, Tyrosine kinases) that may transform a
normal cell into a malignant cell.
 Mutations involving oncogenes involve activation/amplification
14
MCD Genetics
Alexandra Burke-Smith
Tumour Suppressor genes
 Most TS genes require 2 mutations for tumorigenic effect : Hit1 reduces transcript level but insufficient to
cause a phenotypic effect. Requires inactivation of second allele (hit 2), causing total loss of transcription, for
malignant phenotype to be conferred.
 A few TS genes require only one hit: Single hit causes reduction in transcription which for these genes is
sufficient to have a biological effect via “haploinsufficiency”- i.e. reduction in level of protein product
Identification of candidate TS genes
 Using Detection of loss of heterozygosity (LOH)
 Normal tissue: Heterozygous for both polymorphisms
 Malignant tissue: Homozygous (heterozygosity lost through hit 1-point mutation, and hit 2-gene deletion)
Types of Genetic Changes that promote cancer
Altered Levels of
Transcription
Novel aberrant
(abnormal) transcript
“Cytogenetic” changes
“Molecular genetic”
changes
Translocation of a
chromosome segment
Epigenetic changes
Other alteration of
genetic sequence e.g.
internal tandem
duplication (adjacent
repeat of a DNA
sequence)
Deletion of a
chromosome or segment
Point mutation 
frameshift
Gene fusion via
chromosome
rearrangement
Duplication of a
chromosome or segment
Internal tandem
Duplication (ITD)
Down-regulation (TS)
Point mutation
• Mutation in promotor
region
• Truncating mutation
leading to degradation
• Gene deletion
• Epigenetic silencing
(methylation,
acetylation)
• Caused by “2 hits” or
haploinsufficiency
Up-regulation (ONC)
• Gene
amplification
• Activating
mutation
• Influence of new
promotor via
chromosome
rearrangement
Somatic (acquired) vs. inherited changes
• The vast majority of cancer cases (approx 99%) are sporadic caused by the progressive accumulation of new
genetic changes in somatic tissue (“somatic mutations”)
• 1% of cases of cancer are caused by the inheritance from one parent (or occasionally both) of a high-risk
“germline” mutation in a particular gene, usually tumour suppressor
• Why “high risk” mutations?
1) because mutation is recessive, therefore additional somatic hit on other allele still required for
inactivation
15
MCD Genetics
•
•
Alexandra Burke-Smith
2) Could be mutation in DNA repair gene- repair is compromised but still need to wait for somatic mutation
in key TS gene
Predisposition is usually to particular type(s) of cancer in tissue where the function of the gene is particularly
vital
People carrying these mutations are at high risk of developing the associated cancer(s), but overall the
mutations are responsible for only a small proportion of all cases of the cancer.
(for table of mutations- see powerpoint)
Inherited predisposition to breast and ovarian cancer
•
•
•
•
•
•
Inherited mutations in the high-risk breast cancer genes BRCA1 and BRCA2 account for 80 per cent and 14
per cent, respectively, of families with both ovarian and breast cancer.
They only for 2-5 per cent of breast cancer cases overall.
Women who have an abnormal BRCA1 or BRCA2 gene have up to an 60% risk of developing breast cancer by
age 90
Increased risk of developing ovarian cancer is about 55% for women with BRCA1 mutations and about 25%
for women with BRCA2 mutations.
Earlier age of onset than women without inherited BRCA1 and BRCA2 mutations
BRCA2 mutations also predispose to breast cancer in men as well as in women. Around three-quarters of
families with cases of both male and female breast cancer carry mutations in BRCA2. Mutations in BRCA1
and BRCA2 are also associated with increased risks of prostate, bowel and pancreatic cancers
Genetic mechanisms
• BRCA1 and BRCA2 repair double-strand breaks (which may be caused by natural radiation or other
exposures) in DNA in cooperation with other proteins including Rad51, thereby maintaining stability of the
genome.
• The double-stranded break repair mechanism that BRCA1 participates in is called homologous
recombination, in which the repair proteins use an intact sequence from a sister chromatid or homologous
chromosome as a template.
• Hundreds of different types of mutations in the BRCA1/2 genes have been associated with an increased risk
of cancer, including point mutations, several base pair deletions, whole exon deletions and amplifications.
They usually result in a truncated (shortened) non-functional protein
• Inactivation of a second BRCA allele in a cell would generally result in cell death. The rare BRCA-deficient cell
that escapes death to become malignant does so via acquisition of inactivating mutations in other critical
checkpoint (TS) genes, allowing it to proliferate and conferring a survival advantage
Inherited predisposition to bowel cancer
FAP and HNPCC
Two well-known familial bowel cancer syndromes are caused by high-risk mutations in known genes:
1) Familial adenomatous polyposis (FAP), accounts for <1% of all colorectal cancers and is caused by a mutation in
the APC (Adenomatous polyposis coli) gene.
- APC- a classical tumour suppressor gene with a role in cell division via control of the WNT signaling
pathway.
- FAP is characterised by the growth (usually before age of 30) of thousands of intestinal polyps, one or more
of which is likely to become cancerous. Virtually 100% risk of bowel cancer. Average age of onset=39 (65 in noninherited form).
2) Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is more common (3% of cases). It is
caused by a fault in one of a family of DNA repair genes, called mismatch repair genes.
- Mutations in either MLH1 and MSH2 are most common, and account for up to 90% of familial cases.
- Confer a lifetime risk of up to 80% of bowel cancer in men (the risk for women is thought to be lower), as
well as an increased risk of stomach cancer. Women also have increased risks of uterine cancer (lifetime risk of 60%)
and ovarian cancer (lifetime risk of 12%).
16
MCD Genetics
Alexandra Burke-Smith
MYH polyposis
• A more recently discovered less common bowel cancer syndrome, MYH polyposis, is autosomal recessive –
two mutated alleles of the MYH gene (mutY Homologous also known as MUTYH) need to be inherited in
order for an individual to be affected.
• Clinically, MYH polyposis resembles FAP, but the majority of affected individuals tend to have less than 100
polyps (abnormal growth of tissue from a mucus membrane), compared to the many thousands seen in
individuals with FAP.
• MYH is involved in Base excision repair (BER) which protects against damage to DNA from reactive oxygen
species, methylation, deamination, hydroxylation and other by-products of cellular metabolism
Low-risk genetic polymorphisms
•
•
•
•
The tendency of cancers to aggregate in families cannot be wholly explained by rare, high-risk, inherited
mutations.
A substantial proportion of such cancers are thought to be attributable to the combined effects of multiple,
common gene variants, known as polymorphisms, each of which is associated with a small increase in cancer
risk.
A number of polymorphisms that affect the risk of developing different types of cancer have already been
identified. But there are likely to be many more and discovering them is the focus of intensive research.
This search is being facilitated by the availability of the human genome sequence and the development of
high-throughput single nucleotide polymorphism (SNP) array technology
E.g. of a common polymorphism causing cancer: the common colorectal cancer predisposition SNP rs6983267 at
chromosome 8q24 confers potential to enhanced Wnt signalling
Sporadic malignancy: chromosome translocations and oncogenic fusion genes
Our knowledge of the contribution of chromosomal rearrangements to cancer pathogenesis comes from
“cytogenetic” investigations of malignant tissue
This knowledge is most extensive in haematological malignancies (leukaemias and lymphomas) for 2 main reasons
1) Generally leukaemic genomes are more stable than those of solid tumours – therefore easier to pinpoint
pathogenetic changes driving disease
2) Relative ease of performing cytogenetics on haematopoeic circulating cells – easier than with other cells
Chronic Myeloid Leukaemia
• A clonal myeloproliferative (divide uncontrollably without differentiating) disorder of the pluripotent
haematopoeic stem cell
• The type of blood cell that proliferates abnormally originates in the blood-forming (myeloid) tissue of the
bone marrow. It may be acute or chronic and may involve any one of the cells produced by the marrow.
• Blood cells in patients contain a reciprocal translocation between chromosomes 9 and 22, which leads to a
foreshortened long arm of chromosome number 22
• 1 to 2 cases per 100,000
• 15% of all adult leukaemias
• Triphasic (has three phases) - indolent (causing little or no pain) chronic phase, accelerated and terminal
acute stage
• Consistent pathogenomic marker t(9;22),
BCR-ABL
Identification of the BCR-ABL gene fusion
17
MCD Genetics
Alexandra Burke-Smith
Treatment
Interferon-α used to be used, but now Targeted molecular therapy for CML: Imatinib (Glivec) – an effective tyrosine
kinase inhibitor (TKI), which greatly increases survival time from diagnosis. However 20-30% of patients are
resistant/intolerant to Imatinib.
CML treatment is monitored in three ways:
1) Haematologic , e.g. RT/ RQ(real time quantative) PCR
2) Cytogenetic
3) Molecular, e.g. FISH
Sensitivities of the methods used to detect leukaemia in responding and relapsing patients
BM: Bone marrow
PB: peripheral blood
Conventional cytogenetics: Up to 30 metaphases analysed (G-banding)
Disadvantage- relatively low sensitivity, time consuming, only analyse dividing cells
Advantage – robust clinical correlations, detects additional abnormalities (disease progression)
See powerpoint for diagrams based on these methods
Importance of cytogenetics
I.e. why do we quantify outstanding disease in CML?
• Cytogenetic response in first 12-18 months accurately defines response to TKI and helps guide clinical
management
• Absence of cytogenetic response by 12 months – change of therapy indicated
• Degree of cytogenetic response over time is predictive of survival
• Loss of major molecular resistance is indicator of therapy resistance and imminent relapse- change of
therapy indicated
18
MCD Genetics
Alexandra Burke-Smith
Mechanisms of imatinib resistance





A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a protein (tyrosine residue)
in a cell (substrate).
It functions as an "on" or "off" switch in many cellular functions, such as regulating cellular activity including
cell division.
In CML, the tyrosine kinase enzyme ABL is stuck in the "on" position; imatinib binds to the ATP site of BCRABL, locking it in a closed conformation, and preventing the tyrosine kinase activity.
The majority of patient’s resistance coincides with reactivation of the tyrosine kinase activity of the BCR-ABL
fusion oncoprotein. This can result from gene amplification and, more importantly, point mutations that
disrupt the binding of imatinib to BCR-ABL leading to the formation of the open/active conformation of the
oncoprotein.
This confers resistance to the drug, and the DNA mutations in the BCR-ABL oncoprotein are clinically useful
markers of resistance.
Second Generation Tyrosine Kinase Inhibitors
 Derivatives of Imatinib, e.g. Dasatinib and Nilotinib, are used with imatinib-resistant patients
 CCR (all cells normal) or MCR (>65% of cells normal) in up to 60% of imatinib non-responders
 Resistance remains a challenge, e.g. the T315I kinase domain mutation at amino acid 315. This leads to the
change of Threonine for isoleucine, which changes the shape of the oncoprotein, resulting in the open
conformation.
Recurring chromosomal translocations in acute myeloid leukemia
 Most translocations are easy to monitor by cytogenetics and/or FISH
 Molecular diagnosis (RT-PCR) has only been optimised for a subset of these
translocations, usually those which like CML are well controlled by therapy but
not “cured”
Acute promyelocytic leukemia (APML/AML-M3)

Abnormal accumulation of immature granulocytes called promyelocytes
19
MCD Genetics








Alexandra Burke-Smith
Characterized by a chromosomal translocation involving the retinoic acid receptor alpha (RARα or RARA)
gene and the promyelocytic leukemia gene (PML) on chromosome 15, a translocation denoted as
t(15;17)(q22;q12)
RARα/RARA is a member of the nuclear family of receptors; its ligand, retinoic acid is a form of Vitamin A and
acts as a regulator of DNA transcription
Translocation product is PML-RARα fusion protein.
This fusion protein binds too strongly to DNA via enhanced interaction with co-repressor molecules,
blocking transcription
APML is unique from other forms of AML (acute myeloid leukaemia) in its responsiveness to all trans retinoic
acid (ATRA) therapy, a vitamin A derivative.
ATRA dissociates co-repressors allowing normal transcription and cell differentiation
ATRA is not the same as other chemotherapy- it does not kill cells. It is effective when taken continuously
but residual stem cells remain
Like CML, APML is monitored by cytogenetics and/or FISH and/or RQ-PCR
Other examples of genetic markers of sporadic malignancy with diagnostic/clinical applications



Translocations involving the immunoglobulin (IgH) gene are common in lymphoid malignancy (FISH/Gbanding) – partner gene can help determine subtype
Deletion of the short arm of chromosome 5 in myelodysplastic syndrome - Diagnostic marker and predicts
for good response to Lenalidomide (G-banding/FISH)
t(12;21) (ETV6-RUNX1) in childhood acute lymphoblastic leukaemmia predicts for good response to therapy
(FISH/G-banding)
Pharmacogenetic/prognostic:
 P53 (specific gene) deletions in chronic lymphocytic leukaemia and multiple myeloma predict for aggressive
disease and may change patient management (FISH)
 HER2 (ERB2) amplification in breast cancer is a marker of aggressive disease but also predicts efficacy of
herceptin. (FISH)
 T315I mutation in chronic myeloid leukaemia predicts resistance to tyrosine kinase inhibitor therapy (direct
sequencing)
G-banding is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes.
The metaphase chromosomes are treated with trypsin (to partially digest the chromosome) and stained with Giemsa.
Dark bands that take up the stain are A and T rich. This is useful for identifying differences between chromosomes.
20
MCD Genetics
Alexandra Burke-Smith
5. Prenatal Diagnosis
Mr Ruwan Wimalasundera (r.wimalasundera@imperial.ac.uk)
Outline the following:
1. Indications for Prenatal Diagnosis
2. Antenatal Screening for Aneuploidy (Down Syndrome)
3. Prenatal Testing
a. Amniocentesis
b. Chorionic Villus Sampling
c. Fetal Blood Sampling
d. Elective late karyotyping
4. Cytogenetic Techniques
Management options
Indications for Prenatal Diagnosis
I.e. why do we screen women?
All pregnant women in the UK undergo two basic screenings:
1. At 12 weeks: down syndrome screening
2. At 22 weeks: structural anomaly screening
Maternal age is also included in the calculations involved with screening.
However, screening has an important use for women who:

-
Are at high Risk of aneuploidy (chromosome abnormality)
High risk on Down Syndrome screening
Previous aneuploid fetus
Maternal request eg. Age
 Have a known genetic disorder within the family
- Achondroplasia (where the bones of limbs fail to grow to normal size due to a defect in both cartilage and
bone)
- Cystic Fibrosis
- Haemoglobinopathies , eg SCA (sickle-cell)
- X Linked disorder, eg haemophilia
- Parental Balanced Translocation (exchange of genetic material is even so no genes extra or missing)

Have a structural anaeuploidy detected in fetus on routine ultrasound screening
Down Syndrome




-
Most common form of mental retardation in UK (1 in 700 pregnancies), not inherited
Often associated with birth defects- including cardiac, renal, GI abnormalities
Variable severity, not predictable by scan
Due to extra chromosome: Trisomy 21
Standard trisomy 21 - 95% (extra chromosome)
translocation - 4% (tripled genetic material, but only two chromosomes)
mosaic - 1% (mix of normal and mutated chromosome 21- cannot predict severity)
Trisomy: a condition in which there is one extra chromosome present in each cell in addition to the normal diploid set.
 Risk increases with woman’s age due to the fixed number of ova when a female is born. This has an
exponential increase after 35.
21
MCD Genetics
Alexandra Burke-Smith
Down Screening
 Maternal Age
 Nuchal Tranlucency- skin behind neck more “loose” therefore there is more fluid present
 Serum Screening
Can look at biochemical markers (first trimester serum markers) released by the placenta:
- PAPP A
- Unconjugated estriol
- AFP
- Inhibin
- Free beta hCG




Individually these tests have many false positives before diagnosis
Want the highest detection for the lowest false positive rate
Combined test at 10-136 weeks: NT measurement, with free beta Hcg, PAPP A and maternal age
consideration
This is responsible for 90% of down baby diagnosis, with only approx 5% false positives
 Nasal Bones
- 3D fetal ultrasonography is useful for showing parents what the abnormality is
- Some fetuses show complete absence of nasal bone ossification (the synthesis of bone from cartillage),
while others show markedly shorter nasal bones than genetically normal fetuses of the same gestational age
- 85% sensitivity with a 1% false positive rate in detecting aneuploid foetuses
Limitations
- Large population based study show poor utility of measurement
- Racial variation- NB absent in 0.4% of normal Caucasian population and absent in 8.8% of Afro-Caribbean
population
- NB currently of limited value in T21 risk assessment
 Screening Tests
- Triple test
14-21 weeks: AFP, unconjugated oestriol (uE3), and hCG together with maternal age.
- Nuchal Translucency Scan (NT scan)
11-136 weeks: measurement of the fold of skin on the back of the fetal neck (Nuchal Translucency) together
with the maternal age.
- Quadruple test
14-21 weeks: AFP, uE3, free β-hCG (or total hCG) and inhibin-A together with maternal age.
- Combined test
10-136 weeks: NT measurement with free β-hCG, PAPP-A and maternal age.
- Integrated test
Integration of NT measurement and PAPP-A in the first trimester with serum AFP, β-hCG , uE3 and inhibin A
in the second. In the SURUSS study, this was found to have the lowest number of unaffected foetuses lost,
and the highest number of DS diagnoses per unaffected foetus lost.
22
MCD Genetics
Alexandra Burke-Smith
National Guidelines
NICE- Antenatal Care Guideline
April 2005 60% detection for 5% False positive
April 2007 75% detection for <3% False positive
At 1:250 risk cut-off at term
April 2007
Integrated Test
Combined Test
Serum Integrated Test
Invasive Prenatal Diagnosis



Foetal MRA can be used to give more subtle images, with higher resolution, for example can be used to look
at the brain to see the relative movement of white and grey matter.
However invasive prenatal testing is used to sample the foetus, which can provide us with more molecular
information.
Chorionic villi and amniotic fluid can be used to look at the foetal karyotype, DNA analysis and biochemical
analysis
Amniocentesis
•
•
•
•
•
•
•
Performed >15 weeks
Aseptic technique - gloves, no touch
Continuous US guidance
Avoid placenta
22G needle with stylet
Discard first 2ml (so as to only obtain amniotic fluid with the foetal cells)
Aspirate 15-20ml
Complications
 Pregnancy loss rate:
- 1% procedure-related miscarriage
- Only 1 RCT (Tabor 1986), control 0.7% miscarriage, amnio 1.7%
- Fetal Medicine Units 0.5% (1:200)

-
Rh sensitisation
Three Rhesus Protein (Rh; C, D and E)
Antigen on surface of red blood cells
15%of Caucasians are Rh D negative
If the baby is Rh D positive, and the mother is Rh D negative, the maternal immune system will be exposed
to the Rh D on the surface of the red blood cells
The mother will then develop an immune response against the subsequent pregnancy, causing the baby’s
immune system to break down the RBCs and become anaemic
Analysis of Rh can be done by blood test, and all Rh Negative women get Anti D within 72h
 1.3% procedure-related liquor leakage
- Usually self limiting
- Only small number miscarry
 Infection
- <0.1%
- if suspected do repeat amniocentesis, and perhaps suggest emptying uterus
 late diagnosis
23
MCD Genetics
Alexandra Burke-Smith
Early vs Midtrimester Amniocentesis
• CEMAT Trial (Lancet, Vol 351, Jan 1998)
• Early (11+0 - 12+6 weeks)
• Midtrimester (15+0 - 16+6 weeks)
• 4374 women
- Post-procedure loss rate - 2.6% vs 0.8%
- Talipes - 1.3% vs 0.1%
- Culture failure - 1.7% vs 0.2%
- Amniotic fluid leakage (<22 weeks) - 3.5% vs 1.7%
Cytogenetic Analysis
• Fetal cells concentrated in centrifuge (skin, pulmonary, urogenital, extra-embryonic membrane cells)
• Cells cultured in multiple cultures (14 days)
• Culture failure rate 0.5% (1:200)
• Maternal contamination rare- as needs to pass through cells of mother before reaching amniotic fluid,
therefore first 2mls are discarded before aspiration of 15-20mls)
• Human error
• Culture Artifact
• Mosaicism (some normal cells, some abnormal)
Mosaicism in Amnio Culture
 Finding of 2 or more cell lines with different chromosomal constitutions in amniocyte culture e.g.
46XX/47XX+21
 Most often due to culture artefact, therefore has to be present in >2 cultures to be significant (<0.2%)
 As amniotic fluid culture based on various cell types taken from the body, mosaicism is most likely to
represent a true mosaic (exactly which chromosomes are present) in foetus
 However, if fetus structurally normal then may need further confirmatory testing such as fetal blood
sampling as may be confined to fetal membranes.
Chorionic Villus Sampling (CVS)
•
•
•
•
•
•
•
•
11 weeks onwards
Transabdominal (more common) or transcervical (presents higher risk of miscarriage)- USS guided
Needle goes into placenta, and is moved back and forth to obtain culture.
Short term culture gives count in 48 hours- allows for early detection
Ideal for DNA analysis
Tertiary referral unit
Risk of miscarriage 0.5-2%
More complicated technique than amniocentesis
Complications
 Pregnancy loss rate:
- 1% procedure-related miscarriage- not noticeably greater than amniocentesis
- Background miscarriage rate higher 2%
- Fetal Medicine Units 0.5% (1:200)- therefore should only be done in Fetal Medicine Units
 Rh sensitisation
- All Rh Negative women get Anti D within 72h
 Bleeding/ ROM/ Infection rare
- can get vaginal bleeding without miscarriage
 Fetal anomaly, e.g. Limb Defects
- if <10 weeks, limb defects may occur as cells have not fully differentiated (2%)
- 1:1692 background incidence of limb defects
24
MCD Genetics
Alexandra Burke-Smith
- 1:1878 incidence after CVS (>10/40) – therefore increase
For more statistics on CVS vs Amniocentesis etc, look at powerpoint!!
Cytogenetic analysis
 Direct culture exclusively from cytophoblasts/synctiotrophoblast in placenta possible in 72h
 Culture from primarily fibroblasts which are derived from inner cell mass and therefore more representative
of fetus possible in 14 days
 1:500 culture failure
 1:200 Mosaicism - Usually confined placental mosaicism (<10% confirmed in fetus). Mutations can occur in
placenta without occurring in baby. Therefore if CVS is positive, but baby looks structurally normal, we wait
and use amniocentesis.
 0.03% false negative
Fetal Blood Sampling
 Transabdominal USS (ultrasound) guided- done using aseptic technique
 >18 weeks- can be done earlier, but very difficult due to small size of cells
 Asceptic conditions
 CVS and Amnio are preferred for karyotype as they are easier to do
 Primary use if for assessing fetal anaemia, and whether a blood transfusion is reuired
 Transplacental into umbilical cord insertion into placenta
 Or transamniotic into Intrahepatic Vein (within umbilical cord)
Intrahepatic vs Cord insertion
Rapid Cytogenetic Testing
Rapid Karyotyping
 FISH: fluorescence in-situ hybridisation
- used to detect and localize the presence or absence of specific DNA sequences on chromosomes
- chromosome specific
- fluorescence labelled
- uses DNA probes which bind to the specific target sequences
-
 Quantitative Fluorescent PCR
Used to investigate extent of trisomy 21
Any sample can be used to look specifically at
chromosome 21
25
MCD Genetics
Alexandra Burke-Smith
Elective Late Karyotyping
Pros
Avoids risk of miscarriage
Allows antenatal diagnosis
Allows TOP
Cons
Late termination of pregnancy (TOP)
Low utility
Iatrogenic prematurity (induced prematurity by
physician)
Fetal cells in Maternal Blood





Free fetal DNA (ffDNA) is present in maternal circulation (5-10%)
Ff mRNA specifically found in placenta, not in maternal blood
This is cleaved from the circulation within two hours of delivery
Difficult to extract pure ffDNA but can look for specific abnormalities
Clinical use
- fetal RhD genotyping
- sex determination and X-linked diseases
Management Options
If the foetus is diagnosed as positive for Down syndrome, an explanation of the syndrome, as well as its specific
effects on the baby need to be explained clearly to the parents. There are then different things to consider
 Termination of Pregnancy
 Continuation of pregnancy
- Support Parents decision
- Offer continued USS monitoring
- Detailed plans need to be made for
- Mode of Delivery
- Monitoring in Labour
- Neonatal resusitation
- Postmortem
- Postnatal care- surgical/cardiac/neurological etc
 Genetic Counselling
- Risk of recurrence
- Management of future pregnancy
- Implications to other family members
Abortion Act 1967: HFEA 1990
A The continuance of the pregnancy would involve risk to the life of the pregnant women greater than if the
pregnancy were terminated
B The termination is necessary to prevent permanent injury to the physical or mental health of women the
pregnant
26
MCD Genetics
Alexandra Burke-Smith
C The pregnancy has NOT exceeded its 24th week and that the continuance of the pregnancy would involve
risk, greater than if the pregnancy were terminated, of injury to the physical or mental health of the pregnant
women
D The pregnancy has NOT exceeded its 24th week and that the continuance of the pregnancy would involve
risk, greater than if the pregnancy were terminated, of injury to the physical or mental health of any existing
children of the family of the women
E There is substantial risk that if the child were born it would suffer from physical or mental abnormalities as
to be seriously handicapped
Terminations



93% of abortions carried out under clause C & D
<1% are performed > 24 weeks
96% of T21 and Spina bifida performed under clause E
Clause E
There is substantial risk that if the child were born it would suffer from physical or mental abnormalities as to be
seriously handicapped
 This does not give a gestation limit. Most common use is that if there is a 30% chance of handicap this
applies
27
MCD Genetics
Alexandra Burke-Smith
6. Complex Genetic Diseases- Obesity
Dr Alexandra Blakemore (a.blakemore@imperial.ac.uk)
Summary




Common diseases may have a range of causes, some very strongly genetic
Progress in genetics is very fast
Genetic cause does not imply that there’s nothing that can be done
Do not suspend evidence-based medicine because of stigmatisation of particular patients
Introduction to genetics of obesity
1) Syndromic – part of a syndrome eg Prader-willi
2) Monogenic- “single-gene”
3) Common obesity- in the general population: cause unknown
Fat
Necessity
Storage of food and water
Insulation
Support and protection of vital organs
Source of hormones – regulator of reproduction
Sexual signalling
Regulator and fueller of the immune system
Source of new immune cells
Aids wound healing
Not enough fat
Infertility
Miscarriage
Death from infections
Higher suicide rate
Osteoporosis
BMI (Body Mass Index)
Ranges: Underweight, Healthy, Overweight, Obese, Clinically Obese (morbidity)
No real indication of body composition
Doesnt work for very small, or very tall people
Ethnicity: Asians have a lower BMI but more body fat
Body weight is affected by muscle/fat ratio
Obesity
Lack of physical activity + high density calorie diet + stress + genetic factors  OBESITY







Obesity defined as BMI ≥30 kg/m2
Morbid obesity BMI ≥40 kg/m2
Syndromic, monogenic and common forms
In 2005, WHO projected >400 million obese individuals
By 2030, will increase to >1.1 billion obese individuals
Impact on global health; disease e.g. diabetes and costs
Genetics affects individual responses to the obesogenic environment
28
MCD Genetics


Alexandra Burke-Smith
Heritability of obesity estimated at up to 0.77 from twin and family studies (Maes et al 1997, Wardle et al
2008)
All monogenic forms of obesity known so far affect appetite regulation; to gain 1lb of fat requires about
3,500 excess calories
How do we get fat?
 Behaviours
feeding
physical activity
 Physiology
resting metabolism
energy expenditure when active
These are both affected/controlled by genes
Heritability in Children
77% BMI
77% Waist circumference
 Identical twins (monozygotic)
differences all environmental
 Non-identical twins (dizygotic)
have both genetic and environmental differences
Note: the estimated heritability of obesity is approx 30% genetic, and 40% resulting from inactivity, hyperphagia and
unknown factors.
1. Syndromic Obesity






around 30 known syndromic forms of obesity
usually accompanied by mental retardation and particular dysmorphic or clinical features
Prader-Wili syndrome is the most common – imprinting defect, paternal deletion or maternal isodisomy (i.e.
paternal chromosome 15 not inherited)
examination of the underlying mutations can help us to pinpoint previously unrecognised mechanisms of
obesity
also BDNF in WAGR syndrome :Brain-derived neurotrophic factor (BDNF- 11p14.1)has been found to be
important in energy homeostasis, and its deletion results in a predisposition to childhood obesity.
cilia in Bardet-Biedl syndrome: characterized principally by obesity, retinitis pigmentosa, polydactyly, mental
retardation, hypogonadism, and renal failure in some cases. Caused by defects in cell ciliary structure.
2. Monogenic Obesity








Dominant or recessive single gene disorders
Defects in the leptin-melanocortin pathway, which operates in the brain (hypothalamus) to regulate eating
behavior and energy expenditures
Single gene defects found in 1 in 20 morbidly obese children
Most common is MC4R deficiency: autosomal dominant (2-6%)
Leptin: signal produced by fat cells, exported into bloodstream and then transported to the hypothalamus
Overeating leads to severe obesity, fertility problems and immune
Monogenic Leptin Deficiency:
- Immune problems
- Hunger
- Obesity
- no puberty
- poor growth
- low thyroid
this is treated with leptin. However most fat people have lots of leptin- perhaps they just have a
deficiency/loss of function in the leptin receptor in the hypothalamus
29
MCD Genetics




Alexandra Burke-Smith
the leptin-melanocortin ADIPOSTAT regulates body fat levels
- OREXIGENIC: makes you eat
- ANOREXIGENIC: stops you eating
Pro-opiomelanocortin gene: roles in energy homeostasis, pain, melanocyte stimulation and immune
modulation. Mutations lead to:
- Hunger
- Obesity
- Red hair
- Low adrenal activity
PC1 (Prohormone convertase 1), LEP, LEPR, POMC are also single-gene recessive mutations which lead to
obesity
Most people have a mixture of monogenic, oligogenic (two or more), or polygenic mutations contributing
3. Common Obesity









Obesity in the general population
Genomic-wide association (GWA): “common disease, common variant”.
6 GWA studies carried out, looking at single nucleotide polymorphisms SNPs throughout the genome
(comparing obese vs non-obese people)
GWAS-identified SNPs explain only a small proportion of common obesity risk
32 confirmed BMI loci account for just 1.5% of inter-individual variation
Most common marker is FTO- which accounts for a 3kg variation in weight
even meta-analysis involving 0.5 million subjects will only explain 3% of variation in BMI
Problems with GWAS: contribution to the genetic component of BMI is estimated to be low (<5%), so more
work is needed
Also, common variants of more subtle effects (e.g. FTO) is not relevant to sever forms of obesity
Missing heritability?
 Rare variants
 Epigenetics
 Genetic Structural Variations (GSVs):
- Missing pieces of the genome (deletions)
- Extra copies of certain regions (duplications or amplifications)
- Parts of the genome switched around (inversions or translocations)
Ongoing Research



Patients with syndromic forms of obesity have a larger number of GSVs
Investigations of the large number of GSVs found in patients with “obesity-plus” phenotypes to identify new
obesity loci
Then investigate these loci in the general population to find rare variants
Findings: Paper- “The Power of the extreme”
 Chromosome 16p11.2 deletion
 estimated to be 546-700kb
 Probably arises by Non-allelic homologous recombination, implying a size of 739kb
 Using DNA hybridisation, can use markers to compare relative amounts of DNA (normal vs DNA containing
deletion)
 Multiple reports of 16p11.2 deletions and duplications in patients with neurocognitive phenotypes: autism
(0.58%), developmental delay, schizophrenia, bipolar disorder
 Deletion more common in obesity (2.9%)
 Results in the addition of at least 5 BMI points, and is found in 1% of morbidly obese population
 PHENOTYPE:
- BMI = 29.2 kg.m-2 at age 7½ (>97th centile)
- Moderately-severe mental retardation and poor speech
30
MCD Genetics
Alexandra Burke-Smith
-
Dysmorphic features e.g. Congenital nystagmus, squint, hypertelorism, downslanting palpebral fissures,
large protruding ears and bilateral 2+3 toe syndactyly
- Coarse hair with a double crown on his scalp
 Obesity phenotype is age-dependent:
- Adult relatives of probands who also carry the deletion are universally obese
- In carriers of the deletion, the age of onset of obesity appears to be approx. 8-10 years
- In our cohorts, the deletion accounts for:
0.9% of adult morbid obesity
0.4% of child morbid obesity
- 10/25 deletions appear to have arisen de novo(new mutations)
For more information from the study- look at slideshow
 The deletion includes multiple genes that may be candidates for obesity causes
 the first CNV (copy number variation) directly associated with obesity
 Highly penetrant mendelian form of obesity (1 in 20 morbidly obese): i.e. if you have deletion you will get
fat
 RECIPROCAL PHENOTYPE:
Duplications reported to be associated with schizophrenia, bipolar disease and microcephaly
Investigated BMI of 105 duplication carriers, BMI was shifted towards underweight (p=0.04), especially
among adult females (p=0.003) (but not many adult males found)
SUMMARY
Convincing obesity association for deletions of the ~700kb 16p11 ‘autism’ locus
- onset of obesity at 8-10 years of age
- explains ~1% of adult morbid obesity in the general population
- duplication carriers may be more likely to be underweight
Strong association with child obesity of nearby 220kb deletion encompassing SH2B1
- explains >0.5% of child morbid obesity cases
- impact on adult obesity is less clear
- not more insulin resistant than others of similar BMI
Note: Investigation of GSVs in cohorts with “obesity-plus” is a promising route to the identification of novel obesity
loci
Personalised medicine in obesity
Around 1:20 morbidly obese patients has a highly-penetrant Mendelian form of obesity
Obese patients are rarely offered screening or genetic counselling (eg. for obesity or autism risk)
Immediate implications for personalised medicine:
1) Choice of medications that might cause weight gain (especially where there is neurocognitive dysfunction)
2) New drug development (MC4R)
3) Potential for intensive lifelong preventative intervention, e.g. if intervention occurs in childhood, can prevent
adult obesity
4) Choice of obesity surgery type
Effect of MC4R genotype on outcome of obesity surgery
 After 6 years, 16.7% of patients carrying loss-of-function mutations had BMI<30 kg/m2, compared to 42.4%
of subjects with the same initial BMI
 Carriers of loss-of-function or gain-of-function mutations also had higher reoperation rates
31
MCD Genetics
Alexandra Burke-Smith
The future of complex diseases
 Whole genome sequencing
• Millions more SNPs
• Difficult statistical challenges, improvements on use of BMI
 Advanced Phenotyping
• Ultrasound
• Photonic scanning
• Air displacement plethysmography (BodPod)
• CAT/MRI
32
MCD Genetics
Alexandra Burke-Smith
7. The future of Genomic Medicine
Dr Jess Buxton (j.buxton@imperial.ac.uk)
Summary





Summary PGD likely to remain an important reproductive option for a small number of families affected by
serious genetic disease
Pharmacogenetics and identification of disease ‘sub-types’ should reduce side effects and increase efficacy
of treatments
Some companies are already offering genetic tests direct to consumers that have limited clinical utility at
present
Technological advances may one day mean that everyone’s genome is sequenced as part of routine
healthcare
Personalised medicine based on genetic information raises concerns over privacy, autonomy and equality of
access, but also has the potential to transform healthcare
Outline the following:
1. Embryo testing
- Preimplantation genetic diagnosis (PGD)
- Uses and limitations
- Ethical issues
2.
-
Advances in genomic medicine
Pharmacogenetics
‘Next generation’ DNA sequencing
Finding the causes of monogenic disease
3.
-
Personalised healthcare
Direct-to-consumer genetic testing
The personal genome project
Ethical issues
Future perspectives
Embryo Testing
Pre-implantation Genetic Diagnosis (PGD)
PGD: a genetic test carried out on IVF embryos, usually to ensure that only embryos free from a particular genetic
condition are returned to the woman's womb








option for some families at risk of having a child affected by a serious genetic condition
offered as an alternative to prenatal testing
licensed in the UK on a case by case basis, for each new genetic condition that is tested licensing is required
Apr 1990- female embryos chosen to avoid X-linked diseases, adrenoleukodystrophy and X-linked mental
retardation
Aug 2000- first “saviour sibling”- HLA tissue typing for treatment of existing child
Dec 2009- 581 studied babies, conclusion that PGD has no effect on pregnancy outcome up to age of two
months
Over 1000 babies born after PGD worldwide (compared to over 4 million IVF babies), as limited applications
Most common method is blastomere biopsy
1. IVF:
33
MCD Genetics
Alexandra Burke-Smith
collection of egg and sperm, growing of embryo for 3 days to form blastomere (8 identical totipotent
cells)
removal of one or two cells for investigation
2. Test DNA/Chromosomes:
FISH: detects chromosomal conditions, e.g. Down’s syndrome
PCR and DNA sequencing: to detect single-gene mutations
3. Test Results
disorder excluded  implantation
Disorder detected embryo discarded
Ethical issues
 involves discarding unused embryos – this may cause controversy with respect to belief that “life begins at
conception”
 Disability arguments- testing makes judgement on people already living with disability, i.e. their lives are not
worth living etc
 ‘Slippery slope’ – designer babies?
 Eugenics
When is PGD permitted in the UK?
 Severe early onset genetic disease, eg. Tay Sachs
 Severe late onset conditions, eg. Huntington disease
 Disease with incomplete penetrance, eg. hereditary breast cancer (BRCA1/2 mutations)
 To choose a tissue-matched baby for a sick sibling
 Sex selection: not for preference, but in cases of sever X-linked disorders. However sex selection is permitted
in Spain, US
PGD for hereditary breast cancer
 BRCA1 mutation carriers have up to an 80% lifetime risk of developing breast/ovarian cancer, often at a
young age <40
 Birth of baby born following PGD to select embryos free from BRCA1 mutation reignited controversy over
‘designer’ babies last year even though baby’s father’s mum, aunt, sister and grandmother had all been
affected.
Saviour Siblings
 PGD can be used to select an embryo that is both free from disease
 HLA tissue-matched for a sibling affected by a disease that may be treatable using stem cells from umbilical
cord blood, e.g. baby Adam Nash
 Nash case sparked concerns over ‘commodification’ of children into ‘commercial goods’, e.g. “My sister’s
Keeper” etc
Advances in Genomic Medicine
Pharmacogenetics
Studying the genetic basis for the difference between individuals in response to drugs: “right drug, right dose, right
patient”
 People react differently to different medications as a result of variation in metabolism as a result of genetics
 Getting the DOSE right
E.g. Variants in TPMT (thiopurine methyltransferase) gene affect metabolism of 6-mercaptopurine (used to
treat leukaemia)
- Most people metabolize the drug quickly, so doses need to be high enough to treat leukaemia and prevent
relapses
- others metabolize the drug slowly and need lower doses to avoid toxic side effects
- a small proportion of people metabolize the drug so poorly that its effects can be fatal
34
MCD Genetics
Alexandra Burke-Smith
- after a simple blood test, individuals can be given doses of medications that are tailored to their genetic
profile
 Getting the DRUG right
e.g. type 1 diabetes can be misdiagnosed as Maturity onset diabetes of the young (MODY)
Type 1 Diabetes
MODY
Usually childhood onset
Usually childhood onset
Autoimmune disease
Monogenic disease
Must be treated with daily insulin injections
Some types can be treated with oral sulfonylurea
drugs
Advances in DNA Sequencing
 ‘Next-generation’ sequencing already used to identify novel gene mutations in monogenic disease
 Whole exome sequencing (WES) – ie. just protein-coding genes
 Mutated genes involved in Miller syndrome and Schinzel-Giedion syndrome identified in 2010
 In both cases, whole-exome sequencing (WES) carried out on just 4 affected individuals
 Miller Syndrome
- Caused by mutations in DHODH gene
- Multiple malformation syndrome
- Characteristic facial features, including ‘cupped’ ears
- Absent toes
 Schinzel-Giedion Syndrome
- Caused by mutations in SETBP1 gene
- Severe mental retardation
- Multiple congenital abnormalities
- Characteristic facial features
- Life-limiting
 Neonatal Diabetes
- Mutation in ABCC8 gene identified- known MODY/PNDM gene – mutation had been missed previously using
old DNA sequencing techniques
- Single patient with permanent neonatal diabetes investigated using whole exome sequencing
- Patients with this type of diabetes can be treated with oral sullfonylurea drugs, rather than insulin injections
Personal Healthcare
Direct to consumer (DTC) genetic tests
- DTC genetic tests look at particular genetic variance within disease
- Bypasses the doctor-patient relationship
Monogenic Disease
Complex Disease
Can provide carrier status information about
Often have limited clinical utility
recessive diseases e.g. Tay sachs, cystic fibrosis
Can detect rare serious conditions in newborn,
e.g. MCAD deficiency
May cause undue alarm
To determine risk of later onset disease, eg.
hereditary breast cancer
May offer false reassurance
Essential that service includes genetic
counselling
Data privacy concerns
Medium chain acyl CoA dehydrogenase (MCAD) deficiency is an autosomal recessive disorder of beta-oxidation of
fatty acids, which occurs in approximately 1 in 20,000 live births. MCADD generally presents clinically between the
second month and second year of life – can be fatal if undiagnosed, as body cannot combat hypoglycaemia by
producing glucose via fatty acid metabolism
35
MCD Genetics
Alexandra Burke-Smith
DTC for common diseases- Type 2 diabetes
 currently affects 285 million adults worldwide, predicted to rise to 439 million by 2030 due to rise in obesity
 Caused by complex interaction of non-genetic (eg diet) and genetic factors
 Biggest factor is waist circumference
 38 confirmed T2D genes so far, which explain just ~10% of estimated genetic contribution
 Strongest associations are with variants in the TCF7L2 gene , but this is only 1/32 genes
 False reassurance- people who have no copies of the risk variant but will still develop the disease
CONCERNS
 False alarm- people who have at least one copy of the risk variant but will never develop the disease
 Tests of dubious clinical benefit may be offered
 Data protection (especially if company ceases trading)
BENEFITS
 the results of the tests may lead people to make better “lifestyle choices”, which could reduce the risk of
T2D
Whole Genome Sequencing
 Individual genetic tests for variants
 associated with Monogenic disease, drug response, disease “sub-type” and future risk may be replaced by
whole genome sequencing
 All variants (common and rare)will then all be identified in one analysis
COST
 Entire human genome is made up of 2.9 billion base-pairs of DNA
 First human genome cost $3 billion to sequence, now around $10,000
PERSONAL GENOME PROJECT (PGP)
 volunteered to share their DNA sequences, medical records, and other personal information with the
research community and the general public
 project that aims to bring personal genomics into mainstream medicine
The Future: Genetic Profiling?
HGC: Human Genetic Council Conclusions and Recommendations:
 genetic profiling is feasible and likely to become available commercially in less than 20 years
 before the offer of universal genetic profiling could be considered at a population level, steps would
need to be taken to preclude any misuse of information derived from it
 genetic profiling is unlikely to be publicly affordable within 20 years
 for newborn genetic profiling, issues of consent and the welfare of the child are problematic
 genetic profiling may in the future have clinical potential but its effectiveness cannot yet be judged
 there is a pressing need to develop a programme of research to define the full costs and potential
benefits of genetic profiling for the health of children and adults
 Genetic profiling cannot be applied as an NHS screening programme in the near future. The topic
should be kept under review and be revisited in five years
Ethical Issues
 Commercial genetic testing for disease risk
 Equality of access to genetic information
 Right ‘not to know’ (particularly children)
 Protection of data, right to ‘genetic privacy’
36