Lecture 22

Question 1

Genomic Imprinting

Answer 1

Restriction of expression to either maternal or paternal allele (separately/only one) in somatic diploid cells of embryos and adults (RNA or protein) (heteozygoud and homozygous dominant and recessive based on fact that expressing material from both maternal and paternal alleles fairly equally)
Know an epigenetic phenomenon:
-Epigenetics: an epigenetic trait is a stably heritable phenotype resulting form changes in a chromosome without alterations in the DNA sequencing (doesnt depend on changing sequence of DNA bases)
-changes e.g adding methyl groups to the starting points of genes-altering gene expression. Altering histones (proteins around which genes are packed)
Causes different expression from genetically identical alleles
Operates at transcription level
-allele-specific epigenetic modification: e.g cytosine methylation (adding methyl groups to cytosine residues, often placed at start of genes, on cpg islands, which are in promoters of gene)
-phenomenon can be mediated by variety of different enzymes
~0.1% of genes in mammalian genome showing imprinting
(1/1000 genes effected with methalation phenomena only occuring on DNA inherited from one or the other parent)
“imprinting”= parent of origin dependant manner= only occurs to DNA inherited from mother or father, but NOT BOTH

Question 2

Examples of syndromes caused by abnormalities in an imprinted locus: Prader-Willi Syndrome

Answer 2

Features:
failure to thrive in infancy and early childhood
neonatal hypotonia
rapid weight gain after 1 year
behavioural obesity and short stature (partially due to changes in the brain which cause large apetite)
almond shaped eyes
hypogonadism
small hands and feet
skin hypopigmentation
behavioural problems

70-75% have deletion of paternal copy of 15q11-q13
20-25% have maternal uniparental disomy (UPD) chromosome 15

Question 3

Examples of syndromes caused by abnormalities in an imprinted locus: Angelman Syndrome

Answer 3

"happy, laughing child" (happy puppet syndrome)
Open-mouthed expression
Tongue thrusting
Mental retardation
Motor retardation (jerky movements)
Ataxia
Hypotonia
Seizures 
Absent speech
Sleep distrubances
Happy children laughing frequently
Skin hypopigmentation
60% have deletion of maternal copy of 15q11-q13
5% have paternal UPD chromosome 15
a few % have mutation in the PW/AS imprinting centre
deletion and UPD tends to be sporadic
recurrence risk for imprinting centre mutations, translocations and other mutations (e.g. UBE3A)

Question 4

What is the biological difference between Prader-Will and Angelman syndromes which are caused by abnormalities in an imprinted locus?

Answer 4

methylation dependant on parent of origin at region of chromosomes 15 (q11 region)
depending on which parent the methylation has been inherited from
-both have overlapping clinical phenotypes

Question 5

Case study

Answer 5

nonconsaguineious parents= not related (autosomal recessive disorders probably less likely)
Hypotonic-floppy baby
No dysmorphic features= normal looking baby. except Hypoplastic scrotum and cryptorchidism

Question 6

Biological background of Prader-willi syndrome

Answer 6

Overlapping but different phenotype in Angelman syndrome
Both localised by cytogenetic analysis to same region of chromosome 15q11-q13
Aetiology unknown until molecular analysis identified atypical modes of genetic inheritance
Now recognised that distinct by adjacent segments of chromosome 15q11-q13 are critical for normal development
PWS: loss of PATERNAL segment of chromosome 15q-a13
For those genes affects in PWS, the maternal copy is usually imprinted (and therefore silenced) (methylation affecting maternal copy, normal copy turned of by imprinting, therefore relying on paternal copy. therefore get syndrome if deletion or mutation on paternal copy, you’re affected/get syndrome) maternal=inherited as non-expressed gene
FISH demonstrating deletion (del) of SNRPN probe on one of the chromosomes 15s (only 1x yellow marker on paternal, vs 2x yellow markers on silenced maternal chromosome of the pair) (one of the genes, as detected by FISH, has been deleted. Alongside alot of the genes surrounding it)

Question 7

Biological background to Angelman syndrome

Answer 7

Overlapping but different phenotype in Prader-Willi Syndrome
both localised by cytogenetic analysis to same region of chromosome 15q11-q13
Aetiology unknown until molecular analysis identified atypical modes of genetic inheritance
Now recognised that distinct but adjacent segments of chromosome 15q11-q13 are critical for normal development
Angelman syndrome: loss of MATERNAL segment chromosome 15q11-a13
For those genes affected in PWS, the paternal copy is usually imprinted (and therefore silenced)

Question 8

How can Prader-Willi and Angelman Syndrome occur genetically?

Answer 8

Prader Willi= deletion of paternal allele, maternal allele has methyl groups on it due to process of imprinting which has turned this allele off
Angelmans= deletion fo maternal allele and silences paternal via imprinting methyl groups
Other methods to get same result:
1. Uniparental disomy= embryo inherits 2 copies of a locus from one parent and none from the other parent (error in cell division) (P.w. = inheriting 2x maternal copies, both have turning off/imprinting of genes)

2. Imprinting centre(locus/region of genome) mutations or deletions = block imprint switches in the germline (for adding/taking away methyl groups in germline) = Rarer = only have single allele, which has been predisabled

15q11 contains both paternally expressed genes (SNRPN) and maternally expressed genes (UBE3A)
Different phenotypes=different sex alleles, imprint different genes

Question 9

Genetic imprinting: role of DNA methylation

Answer 9

Methylation of DNA in mammalian cells occurs at cytosine residues in CpG islands
-methylation allows imprinting
-methylation major mechanism for regualting gene expression, core of alot of epigenetics
CpG island = >200 bp regions of DNA with G + C >0.5 located mainly in promotor regions
CpG methylation of promotes causes transcriptional silencing
CpG methylation transmitted through cell divisions by maintenance of (enzyme) methyltransferase activity
-methylation can be reversed if methyltransferase inhibited or sequestered (as occurs in early development)- (some drugs can also turn these enzymes off)
Gamete-specific methylation mediated by gamete specific proteins (work with only either male or female gamete) binding imprinted genes during gametogenesis and early embryogenesis

Question 10

Application and removal of imprint

Answer 10

Female: imprinting established during oocyte maturation
Male: imprinting established prior to meiosis in post-mitotic primary spermatocyte
Demethylation occurs in early embryo
primordial germ cells remain unmethylated
may vary between tissues and during development -e.g. maternal-specific expression of UBE3A in regions of brain

Question 11

Epigenetic Phenomenon

Answer 11

-Epigenetics: an epigenetic trait is a stably heritable phenotype resulting form changes in a chromosome without alterations in the DNA sequencing (doesnt depend on changing sequence of DNA bases)
-changes e.g adding methyl groups to the starting points of genes-altering gene expression.
- Altering histones (proteins around which genes are packed)
Causes different expression from genetically identical alleles
Operates at transcription level
-allele-specific epigenetic modification: e.g cytosine methylation (adding methyl groups to cytosine residues, often placed at start of genes, on cpg islands, which are in promoters of gene)
-phenomenon can be mediated by variety of different enzymes

Question 12

Epigenetic Summary

Answer 12

Epigenetics are changes to the expression of genes that are hertiable but not encoded in sequence of DNA

• instead are due to additional of methyl groups to cytosine residues in promotorss (start of genes)
• these phenomena can be mediated by a variety of different enzymes

Question 13

Imprinting summary

Answer 13

When epigenetic changes occurs in a Parent of origin dependant manner
-only happens to the DNA from one (either mother or father) but not both

Question 14

methylation application and removal summary

Answer 14

have eloved CpG islands on many of our genes
-allow methyl groups to be applied to cytosine residues
-methyl groups also applied to cytogene residues in other regions of genes but specifically in promotor region of gene - regulates how much genes are expressed
-wide spread mechanisms = epigenetic mechanism = can turn genes on or off
-important in effect of some environmental or nutritional changes
-also important in inheritance of imprints
imprints = methylation of genes in parent of origin dependant effect
-can lead to PW or A syndrome, dependant on which parent the imprinted gene was, and requiring loss of the same genetic region of the other parent (the only parent that could result in gene expression because first parent had imprint)

Question 15

Methylation changes are also associated with cancer

Answer 15

Methylations= turn off genes
-methylation of tumour suppressor genes
• Inherited predisposition
• Somatic methylation changes

Question 16

Methylation changes are also associated with cancer: Inherited predisposition and

Answer 16

Inherit one copy of a gene from one parent that has methylation
-may turn off Tumour suppressor gene
-if you get damage to copy inherited from other parent (inherited or somatic mutation)
= None of the tumour suppressor gene
=increasing the probability that a tumour will develop

Question 17

Methylation changes are also associated with cancer: • Somatic methylation changes

Answer 17

Have methylation turning on a tumour

• inherited from one parent a mutation in a gene (BRCA1 mutation)
• most cells fine as have other normal functioning wild type other BRCA1 allele. acts as an inhibitor to cells dividing once being damaged in DNA
• if methylation occurs in remaining normal allele = turns off BRCA1 = cells can develop into a tumour

Question 18

Inherited predisposition: eg. Beckwith- Wiedemann syndrome (Chr 11p15)

Answer 18

Chromosome 11 (Chr 11p15)
•Pre and postnatal overgrowth condition
• Macroglossia (large tounge), organomegaly
hemihypertrophy (a symetric effect on different sides of body)
•Increased tumour predisposition (e.g. Wilms tumour)
• Most cases: excess paternally expressed growth enhancer (IGF2) and/or deficiency of maternally expressed growth suppressor (H19 or CDKN1C)
-inheriting methylation of some of the genes which normally prevent tumours growing

Question 19

Inherited predisposition: Other disorders with imprinted genes / parent-of-origin effects

Answer 19

• Wilms tumour: : loss (relaxation) of imprinting at IGF2 locus leads to reduced p57 KIP2/ CDKN1C expression
-combination of methylation of several genes in one region
-CDKN1C regulates cell cycle and response to DNA damage
• Familial paraganglioma

Question 20

Somatic methylation changes in cancer example

Answer 20

CCND1
-gene which encoded for a cyclin (protein which drives cells through mitotic cell cycle)
-higher expression of this gene the more rapidly this cell can divide (a feature of tumour growth)
-see how tumours turn on and off genes
-coloured according to how strongly a particular gene is expressed
methylation vs copy number (inherited extra or less copies of a gene)
-high numbers of extra copies = high expression fo gene
-high amounts of gene methylation = this gene is turned off by methylation in some tumours = slow growing tumours= less Cyclin D1 expression

Question 21

Methylation in tumours

Answer 21

in tumours methylation of somatic cells is very important in evolution of a tumour
-occurs in promotor regions
-often works in synergy with other genetic changes that occur in tumours
-e.g. gain of extra copies or mutation
=somatic methylation with extra copies and mutation

Question 22

Therapeutic inhibition of DNA methyltransferase: Low doses of 5-aza-CR
5-aza-CDR

Answer 22

Demethylation

• 1. Gene reactivation. growth inhibition –> Tumour cells (aberrent hypermethylation)
     
     2. Epgenetic Reprogramming –> No self renewal of tumour intiiating cells
     3. Tolerated –> Bone marrow

Question 23

Therapeutic inhibition of DNA methyltransferase: High doses of 5-aza-CR
5-aza-CDR

Answer 23

Cytotoxicity

1. Kills Tumour cells
2. Transient Block? of tumour initiating cells
3. Toxicity of bone marrow

Question 24

Dynamic Mutation

Answer 24

•Progressive expansion of repeat sequences, with every round of inheritance; mainly
triplet repeats
-if in encoding region of gene, repeating pattern of three bases pairs, will result in repeating tract of same a/acid in a protein
-inheriting more triplet repeats with each generation
• ~ 20 neurological and muscular diseases involving repeat expansion:
-errors during cell division addition extra divisions
-Huntington disease
-Myotonic dystrophy
-Cerebellar ataxias
-Fragile X disease A
Symptoms in later generations often occur at younger ages and a more severe, a phenomenon known as anticipation

Question 25

Case Study 2
A 45 year old man presented initially with declining memory and concentration. As his intellectual function deteriorated during the ensuing year, he developed involuntary movements of his fingers and toes as well as facial grimacing and pouting. He was aware of his conditionandbecamedepressed. Hehadbeenpreviouslyhealthy and did not have a history of any similarly affected relatives; his parents had died in their 40s in an automobile accident. He had one healthy daughter.

Answer 25

After an extensive evaluation, the neurologist diagnosedHuntingtondisease(HD). This was confirmed by DNA analysis showing 43 CAG repeats in one of his HD alleles (normal ≤26).
-duaghter has inherited patten of large number of repeasts (43 repeasts of CAG)
Subsequent presymptomatic testing of his daughter showed that she had inherited the mutant HD allele
Clinical diagnosis diagnosed by George Guntington in 1872

Question 26

Diagnosis of Huntington disease suspected clinically if:

Answer 26

• Progressive motor disability involving both voluntary and involuntary movement
• Mental disturbances including cognitive decline and/or changes in personality
• Family history consistent with autosomal dominant inheritance
-extra copies= producing protein that can dominate over remaining normal inherited allele

Question 27

Pathology of Huntington disease

Answer 27

brain

-loss of mass on RHS

Question 28

Identification of Huntington disease gene

Answer 28

1983
Scientists discover a gene marker linked to HD on the short arm of chromosome 4, which indicates that the Huntington gene is also located on chromosome 4. Predictive linkage testing is introduced to assess the likelihood of having inherited the HD gene.
1993
The location of the Huntington gene is discovered at 4p16.3 on chromosome 4. The gene is found to contain codon C-A-G in varying numbers. An abnormal number of CAG repeats turns out to be a highly reliable way to tell whether someone has the allele for HD.
1993: Pinned down to Huntington gene

Question 29

Allele sizes

Answer 29

Normal: ≤ 26 CAG repeats
no clinical effects, stable in transmission
Intermediate: 27 - 35 CAG repeats
no symptoms of HD, but possibility of child inheriting “mutant allele” if parent male (inheriting repeats plus a few more)
Mutant: ≥ 36 CAG repeats
reduced penetrance alleles: 36 - 39 repeats full penetrance alleles: ≥ 40 repeats
-develop huntingtons yourself later in life

Question 30

Molecular genetic pathogenesis

Answer 30

Protein encoded by HD gene known as “huntingtin”
? normal function: interacts with proteins involved in transcription, cell signalling
and intracellular transport
(gaining of) CAG repeats result in polyglutamine expansion Effect on neuronal cells through gain-of-function
-cannot be compensated from remaining normal allele

Question 31

Molecular genetic testing: clinical method: Targeted Mutation Analysis

Answer 31

Targeted mutation analysis
• PCR-based methods detect alleles up to ~ 115 CAG repeats
• location of PCR primers critical for accuracy of repeat size
• (older anyalysis for smaller numbers) Southern blot sometimes used for large expansions associated with juvenile-onset HD (which may fail to amplify well by PCR) and confirmation of “homozygous normal” genotypes

Question 32

Genotype-Phenotype Correlation

Answer 32

There is a colleration between number of repeats in genome and onset of phenotype
Age of onset vs Number of Repeats
-very low number of repeats dont have any onset
-very high age of repeats = very LOW age of onset
-intermediate number of repeats = progressively older age when disease occurs
-anticipation: when disease becomes more severe and manifests at a younger age, as generations progress
-due to addition of more repeats per generation?
Adult onset: 36-55 CAG repeats Juvenile onset: > 60 CAG repeats
Not a perfect correlation

Question 33

Anticipation

Answer 33

• Increasing disease severity or decreasing age of onset observed in successive generations
• Occurs more commonly with paternal (father) transmission (in Huntington’s) of mutant allele instability of CAG repeat during spermatogenesis (extra copies gained)
• Children with juvenile-onset disease usually have expanded allele from father

Question 34

Risk to family members

Answer 34

Parents of proband
• most individuals with HD have an affected parent
• de-novo mutations causing HD are rare
• asymptomatic father of an affected individual may have
an intermediate allele (intermediate number of repeats)
• either asymptomatic parent may have a reduced penetrance allele (CAG repeat 36-39)

Sibs of proband
• risk depends on genetic status of parent

Offspring of proband
• each child of individual with HD as a result of heterozygosity for the HD mutant allele has a 50% chance of inheriting the mutation
+ added complexity of anticipation phenomena - if inherited through dad may have been slippage of replication of DNA, leading to more repeats, leading to (anticipation) more severe and earlier disease than father

Question 35

Molecular genetic testing

Answer 35

Diagnostic (confirmatory)
• confirms disorder
• undertaken after assessment by neurologist
Predictive (presymptomatic)
• confirms mutation that confers high risk of disorder prior to onset of disease (risk to develop later in life)
• only undertaken after neurological assessment and counselling (essential)
Prenatal
• issues regarding disclosure of status of parent
• exclusion testing (affected grandparental allele)

Question 36

Genetic Counselling

Answer 36

Huntington’s disease raises a number of complex issues related to counselling and ethics
Asymptomatic at-risk adults:
• testing does not provide definitive prediction of age-of-onset or severity
• likelihood that CAG repeat size may cause disease by certain age may be useful
• may seek testing for personal decisions regarding reproduction, financial matters, career planning or simply “need to know”
• requires pre-test interviews to assess motives, knowledge, impact of positive and negative results, neurological status
• counselling regarding potential problems: -insurance, employment, educational discrimination -changes in social and family interactions
• implications for at-risk status of other family members
• requires informed consent and confidentiality of records
• arrangements for long-term follow-up

Question 37

Genetic Counselling extra

Answer 37

three generations alive
child develops the disease but parent hasnt developed the disorder
-does the parents have a right not to know
-by testing the child, what does that tell you about the parent
-grandparent?
-indirectly getting information about the family
Underlines important role of genetic counselling