Genetic Diagnosis, X-inactivation and Hardy Weinberg Equilibrium

Question 1

What are dominant-negative mutations?

Answer 1

Product interferes with function of normal allele product
Heterozygotes show more severe phenotype than LOF mutation
Might be interacting with a partner protein e.g. binding in an incorrect way or different place

Question 2

What is Haploinsufficiency?

Answer 2

Loss of function with dosage sensitivity
Single copy of a gene is not sufficient enough to produce the normal function or phenotype
Dominant Inheritance

Question 3

What are nonsense mutations?

Answer 3

Mutations that produce a premature termination codon
Small indels that are not a multiple of 3 can result in a frameshift e.g. Dystrophin in DMD
Usually result is no protein produced at all rather than a truncated protein as mRNA is unstable and degraded rapidly

Question 4

How can point mutations affect mRNA splicing?

Answer 4

Splice donor (SD) and Splice acceptor (SA) sequences conform to the GT-AG rule - mutation at one of these 4 sites means the splice site won’t be used
Mutations nearby might affect efficiency of splicing - less normal or change in balance between different splice forms
Point mutations may change intronic or exonic sequence into splice sites, resulting in mis-splicing

Question 5

How do mutations cause Cystic Fibrosis

Answer 5

1000 mutations for CFTR reported but F508 is the most common (3bp deletion)
Maintains reading frame but lose phenylalanine
Isoleucine codon (507) changes from ATC to ATT (deletes C of Isoleucine and TT of Phenylalanine)
European 508 frequency increases from southeast to northwest
CFTR is member of ABC transporter protein family in the lungs - cAMP mediated chloride
most mutant CFTR degraded
homozygous most severely affected
Healthy lung: epithelial cells covered by fluid layer and mucus layer with cilia transporting mucus to airway opening
CF lung: Defective Cl- secretion and Na+ hyperabsorbtion deplete airway surface fluid, buildup of more viscous mucus which doesn’t shift bacteria away - increased infection

Question 6

How is Phenylketonuria (PKU) caused?

Answer 6

Enzyme Phenylalanine hydroxylase (PAH) normally converts Phe to Tyr
LOF causes buildup of Phe and secondary buildup of phenylketones which interfere with CNS development
No cure but can decrease amount of Phe coming in through the diet

Question 7

How is Achondroplasia (ACH) caused?

Answer 7

97% have same DNA change (c.1138G>A) creates aa change Gly380Arg which activates FGFR3 (Fibroblast growth factor receptor 3) signalling
Mutation in transmembrane domain > increases stronger dimer formation > increases signaling > reduction is ossification of chondrocytes > lack of bone/cartilage formation
Unaffected fathers in 50s are 10x more likely to have a child with de novo mutations than men in 20s

Question 8

Describe Osteogenesis Imperfecta as an example of the dominant negative effects of missense mutations.

Answer 8

Type 1 procollagen made up of 3aa chains encoded by 2 genes COL1A1 and COL1A2
Null mutations in either result in mild OI (1/2 normal and 1/2 surplus alpha2 chains which are degraded)
Missense produce severe OI (1/4 normal and 3/4 abnormal procollagen)

Question 9

Alpha thalassaemia as an example of a disease arising from rearrangements.

Answer 9

Deletions at alpha-globin locus are frequent and occur due to unequal crossover in meiosis
Normally have 2 alpha-globin genes and one inactive pseudogene
Sequences of DNA adjacent to the gene are very similar so chromosomes can misalign - resulting C have either 1 or 3 functional genes
Disease severity related to gene copy number

Question 10

Charcot-Marie Tooth Disease as an example of a genetic disease caused by rearrangements

Answer 10

Normally have 2 copies of PMP22
Incorrect recombination leads to 1.5Mb duplication
Hetero = 3, homo = 4 (more severe)

Question 11

Describe Haemophilia A as an example of a genetic disease caused by a chromosome rearrangement.

Answer 11

Due to mutations in F8 gene on Xq28 which encodes Factor VIII
Inverted repeats can mispair and cause looping of DNA which then inverses the middle DNA

Question 12

Describe Duchenne muscular dystrophy (DMD) as an example of a genetic disease caused by a rearrangement.

Answer 12

Mutated Dystrophin gene on Xp21 (breakpoint)
Rarely affects females - all carry balanced X-autosome translocations
Girls that are affected is due to preferential inactivation of normal X
Most due to deletions - frameshift and PMC
44 is site of breakpoint in 30% of cases
Dystrophin protein inner face of sarcolemma, linking cytoskeleton to external BM (maintains muscle stability)
No dystrophin protein produced
Can treat with dystrophin, shouldn’t see as foreign

Question 13

Becker Muscular Dystrophy (BMD)

Answer 13

Similar clinical presentation to DMD and mapped to same gene but less severe with later onset
Indels maintain reading frame so dystrophin is just abnormal (usually truncated)

Question 14

Describe dynamic mutations

Answer 14

Short repeated DNA arrays often trinucleotide repeats (CTG, CAG, CCG)
Cause disease when array expands above a critical number
Can be caused by expansion by slippage during replication - one or more bubbles up and re-anneals - can be rapid expansion between generations
coding or non-coding region
Expanded repeats are meiotically and somatically unstable

Question 15

Describe Fragile X Syndrome (FXS) as an example of repeat expansions in non-coding regions.

Answer 15

X linked, males most commonly affected
Some cells show fragile site on X chromosome but not reliable test especially carrier females
Run of tandemly repeated CGG
Tend to grow as repeat as transmitted down generations (anticipation)
FMR1 gene, CCG in 5’ UTR so in mRNA but not translated
<55 stable, non-pathogenic
>55 unstable, 1/3 males develop neurodegenerative syndrome after age 50
>200 classic FX phenotype, silences gene
Methylation and silencing of promoter
Loss of protein in FXS associated with problems in transport and translation of many mRNAs and abnormal synapse development

Question 16

Myotonic Dystrophy 1 (DM1) as an example of repeat expansions in non-coding regions.

Answer 16

Autosomal dominant inheritance with anticipation
Mutation in DMPK last exon (CTG repeat) on chromosome 19
100-1500 classical, many affected organs
>1000 Congenital only seen when transmitted by mother
Expanded repeats trapped in nuclear foci which lead to an imbalance of splicing factors and mis-splicing of many pre-mRNAs

Question 17

Describe Myotonic Dystrophy 2 (DM2) as an example of repeat expansions in non-coding regions.

Answer 17

Expansion of 4bp repeat (contains CTG)
Same Clinical symptoms of DM1
Expanded repeats trapped in nuclear foci which lead to an imbalance of splicing factors and mis-splicing of many pre-mRNAs

Question 18

Describe Huntington’s disease as an example of repeat expansions in coding regions.

Answer 18

Expansion of CAG repeat on C4p
Expansion rarely goes above 100 - affect total protein
Polyglutamine tract protein is in some way toxic to neurones, leading to late onset neurodegenerative disease

Question 19

What are Epigenetic Effects?

Answer 19

Genetic changes that are heritable from cell to daughter cell but don’t depend on primary DNA sequence changes
Associated with DNA methylation
Important in development, X-inactivation and imprinting

Question 20

What is mammalian DNA methylation?

Answer 20

Methyl group added to 5 position of some cytosines to form 5-methyl cytosine (5MeC which can still base pair with guanine same)
Almost entirely restricted to CpG nucleotides
Patterns are inherited in cell division (DNMT1 is responsible for maintenance of meth) - other DNA methylases carry out de novo methylation
Meth pattern erased in early embryo and re-established at implantation, followed by specific
alterations in meth (X-inactivation, de novo methylation and repression of genes necessary for pluripotency) – some sequences escape this de- and re-meth process (e.g. imprinted genes)
Silence, repress, control expression of particular genes at specific dev stages – plays a role in gene dosage effects and the very small subset of particular genes which you regulate based upon whether you’ve got the maternal or paternal copy

Question 21

What is the process of X-inactivation?

Answer 21

Random silencing of either maternal or paternal X chromosome, occurs separately in individual cells during embryonic development
XIST gene expressed from inactivated X and produces mRNA which coats chromosome
Only expressed in cells contaning at least 2 X chromosomes
Silenced X forms dense Barr body and most of the genes are silenced
Cells descended from these will maintain same pattern of inactivation
E.g. Calico Cats - If a female cat is hetero (XtXb), she will have alternating patches of black and tan due to random inactivation of the alleles in each cell
Human females are also mosaics but less easy to see

Question 22

What are some examples of X chromosome aneuploidies and how do they arise?

Answer 22

Turner’s Syndrome:
- Females lose part or whole of X leaving with one normally of maternal origin
- Mild effects as have normal dosage of X
- Develop as females but have some defects (short stature, infertility, learning defects)

Klinefelter Syndrome:
- Males have XXY (or more Xs)
- Will neutralise extra X in same way as females
- May be infertile, less dense body hair

Triple X Syndrome:
- Females with XXX genotype
- Typically fertile but some issues with muscle tone, learning difficulties, late motor development
- 2 XX silenced into Barr Bodies

Question 23

What is genomic imprinting?

Answer 23

Epigenetic phenomenon - difference in expression of alleles according to parent of origin (around 100 genes)
Differentially marked maternal or paternal alleles (chemical markers added during sperm/egg production - if it is imprinted it is silenced)
Differential expressed after fertilisation
If active allele is mutated, leads to disease phenotype
Reset during gametogenesis so correct parent is marked

Question 24

Explain how the same mutation can cause Angelman Syndrome (AS) and Prader-Wili Syndrome (PWS) which have vastly different phenotypes.

Answer 24

Deletions in chromosome 15q11-13
PWS caused by paternal deletion
No single gene mutation
Severe neuroendocrine disturbance
Difficult to assign genes, detect and treat

AS caused by maternal deletion
LOF mutation in UBE3A responsible for <20%
UBE3A encodes ubiquitin-protein ligase, adds ubiquitin to proteins, targeting them for degradation by proteasome
Expressed in brain - hippocampal neurons and cerebellar Purkinje cells
Mice lacking UBE3A show neurological defects

Question 25

Describe mitochondrial mutations and diseases

Answer 25

Genome only 16.6kb but higher mutation rate
Inherited from mother (egg cell)
Heteroplasmy = mixed population of normal and mutant genomes within same cell (extent can vary between mother and child) and can make it difficult to predict severity for child
e.g. Cytochrome c oxidase assay where brown stains positive, blue stains negative cells
Mito gen important role in energy production

Question 26

Which tests are good to test for Aneuploidy?

Answer 26

Array hybridisation: fluorescently label patient and control sample, plot signal, expect specific ratio e.g. increased signal across whole of C21 is Down’s Syndrome
FISH: non-dividing cells, good for prenatal diagnosis, expect 2 copies of control probe and detect number of copies of other C
STRs: Amplification of region on specific chromosome that has STRs

Question 27

Which tests are good for detecting Large Scale Genomic Rearrangements?

Answer 27

FISH and Array Hybridisation (but won’t see balanced translocations for AH)
Identify gain or loss of chromosomal regions - extra shows as dots above line, loss is opposite

Question 28

What tests are good for detecting Specific Deletions And Duplications?

Answer 28

FISH: e.g. VCFS - control probe is inside same chromosome, expect to see 2 copies of control and 2 of target for normal C
MLPA (Multiplex Ligase-Dependent Probe Amplification):
Use specific probe pairs ligated together in presence of template (with stuffer sequence)
Primers bind to sequences, amplify, get product
DNA Ligase covalently link probes together
Denaturation and hybridisation, ligation, PCR with primers, fragment analysis
can change size of stuffer sequence to probe for lots of different sequences at once
Can be used to find exonic deletions and duplications as well as unbalanced translocations (subtelomeric probes)

Question 29

Which tests are good for detecting Triplet Repeat Disorders e.g. Huntington’s Disease?

Answer 29

Polymerase Chain Reaction
Length of PCR product varies according to the number of CAG codons
Expect to see 2 bands with potentially differing numbers of repeats

Question 30

Which tests are good for detecting Known Point Mutations e.g. Sickle Cell Disease and Cystic Fibrosis?

Answer 30

Amplification Refractory Mutation System (ARMS-PCR)
Useful where frequency of a mutation is high
SCD: pGlu6Val mutation in beta globin
1st primer anneals to both normal and mutated allele; 2nd primer specific for normal allele (A) or sickle cell mutation (T); two separate reaction; Look for PCR product for each one
CF: F508 mutation
Multiplex with ARMS essay - can probe for multiple different loci and use different primer pairs all in one reaction (12 but none others)

Question 31

Why is it difficult to find an unknown nucleotide substitution mutation in a patient?

Answer 31

• Large number of mutations are family-specific
• Identification of mutation may require sequencing of entire gene - may lie within introns or regulatory regions
• Many are genetically heterogeneous (mutations in diff. genes can give same phenotype so many might need to be sequenced)

Question 32

Describe the difference between Chorionic villus sampling and Amniocentesis as methods for prenatal diagnosis.

Answer 32

CVS: 1st trimester, small risk of miscarriage, needle and syringe into placenta
A: 16 weeks gestation, fetal cells, small risk of miscarriage, needly and syringe into amniotic fluid
Both need FISH or similar to analyse

Question 33

Describe tests to develop risk factor for trisomies before invasive testing.

Answer 33

Increased maternal age (low sensitivity)
Ultrasound (nuchal translucency - more fluid behind back of baby’s neck suggests developing abnormalities like in trisomies)
Chemical markers in maternal serum (including CGP)
If combined risk is above threshold, offered further testing e.g. invasive or NIPT

Question 34

Describe non-invasive prenatal testing (NIPT).

Answer 34

Obtain maternal blood with contains cell-free fetal DNA (cffDNA) from placenta
Used for diagnosis of single gene disorders

Question 35

Describe Preimplantation Genetic Diagnosis (PGD) including advantages/disadvantages.

Answer 35

Obtain oocyte and fertilise in culture using IVF
Take cell once it gets to 6-10 cell embryo
Offered in UK if there’s previous GD history
A:
>600 approved, more needs application
D:
Technically challenging
Limited material you can obtain
Usually requires exact mutation in family to be known

Question 36

Mendelian diseases: Why are mutations not removed by selection?

Answer 36

Very high mutation rates (recurrent deletions), large gene (DMD), sperm selection (achondroplasia)
Propagation of non-pathological premutations (Fragile X, myotonic Dystrophy)
Onset of symptoms after reproductive age
Hetero advantage (SCD, Thalassaemia)

Question 37

What are founder effects?

Answer 37

Population deriving from a small number of individuals
Any recessive allele present in one of the founders is likely to be present at a high frequency in the modern population

Question 38

Describe methods of screening for Cystic Fibrosis.

Answer 38

Population: Sequencing of gene not yet feasible but PCR-based ARMS tests would miss too many carriers
Neonatal: Universal newborn screening, early diagnosis improves prognosis
Immunoreactive Trypsin:
Pancreatic ducts are blocked, preventing trypsinogen reaching intestine and builds up in blood
Biochemical test measures levels of T in blood (involves antibodies) - raised level indicates follow-up necessary
Prick heel and make blood imprints on Guthrie card

Question 39

Describe newborn blot screening for Phenylketonuria (PKU), Sickle Cell Disease and Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCADD).

Answer 39

PKU: Measure Phe/Tyr ratio using mass spectrometer (Tyr should normally lower)
Aim to start low protein diet plus supplements before 21 days after birth
SCD: Can run blood on electrophoresis gel, separating Haemoglobin based on charge
MCADD: Test by mass spectroscopy - elevated C8 FA (octanoylcarnitine) indicates further testing including genetic screening required

Question 40

Describe what the typical output from Sanger sequencing will look like.

Answer 40

In dominant disease, patient hetero for mutation will appear as N on sequence trace
Harmless variants will too
In recessive disease, patient may be homo for mutation or carry 2 different mutations (one in each allele, compound hetero)

Question 41

Describe Gene Sequencing for PAH (mutations cause PKU).

Answer 41

21 exons spread over about 80kb
As it’s recessive, need to find 2 inactivating mutations to account for features
PCR exons and surrounding bases, sequence
May need to determine copy number of exons, using MLPA
Most common mutations are splice or missense
Compare known disease-associated change in locus specific database to mutation patient has
Can predict effect and see if it will come to disease outcome using prediction software

Question 42

Gene Panels

Answer 42

If there are several possible genes involved, analyse all exons of all these genes
Increasingly done by specific exon capture and NGS (also allows analysis of copy number)

Question 43

Describe the process of exome capture

Answer 43

Construct shotgun library using genomic DNA of patient
Shear, break into fragments of DNA (some are exons, introns, repetitive regions)
Use probes which are against all the different exons - also attached to beads (magnetic)
Probes hybridise to exonic sequence in disease
Pull down the ones we want and wash away what we don’t, enriching captured DNA
DNA sequencing, align against genome and identify where variations in particular exonic sequences can be used.

Question 44

Describe Kabuki Syndrome as an example of whole exome sequencing.

Answer 44

Caused by MLL2 mutations
Some evidence of autosomal dominant
Assumed mutation was rare and missense/nonsense/indel/splice site
Identified variants and compared other
Suggested syndrome caused by haploinsufficiency for KMT2D function
(lysine-specific histone methylase)

Question 45

What does the Hardy Weinberg Equilibrium state?

Answer 45

In an ideal population with no external influences, the relative proportions of different genotypes remain constant from one generation to the next

2 alleles A and a
A occurs at frequency of p, a occurs freq q
Therefore p+q=1
AA = p2, Aa = 2pq, aa = q2
Therefore p2 + 2pq + q2 = 1
Simple representation is Punnett square between two hetero parents

Question 46

What are the influences on Hardy Weinberg?

Answer 46

Non-random mating: assortive mating and consanguinity will skew (favoured genotypes increase in prevalence)
New mutation entering gene pool
Selection for particular genotype (one genotype becomes less prevalent)
Small population size (genetic drift, new population forms from subset of genotypes in original population)
Gene flow (e.g. migration - genotypes that leave become rarer)