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Flashcards in Genomics Deck (172)
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1
Q

What is genomics?

A

The study of whole genomes including their mapping, structure, function and evolution.

This typically refers to changes in DNA sequence.

2
Q

What are regulatory and/or functional genomics?

A

These typically refer to changes in the structure, packaging and function of DNA sequence and DNA products.

3
Q

What does the term Omics mean?

A

Describes the characterisation and quantification of biological molecules in an organism e.g. proteome, microbiome.

4
Q

What is the 100K genomes project?

A

A project aiming to sequence the genomes of patients to help improve disease diagnosis and treatment. Diseases include cancer, rare diseases, infections and controls. 100k+ genes have been sequenced so far. It is a NHS transformation project.

5
Q

What are the characteristics of X-linked recessive inheritance?

A
  • Mutations in one copy of a gene are necessary to cause the disease in males, but females would need both copies of the gene.
  • No male to male transmission.
6
Q

What is genetic linkage?

A

Because recombination can occur at any location along the chromosome, the frequency of recombination between two locations depends on their distance. As a consequence DNA regions that are located in close proximity are more likely to be co-inherited (not split apart) than DNA regions originating from further apart.

7
Q

How can you determine how close genetic loci are to each other?

A

By observing recombinant events. The fewer the recombinant events between loci across generations, the closer they are to each other.

8
Q

What information does genetic linkage provide?

A
  • Helps determine the genotype of a disease causing mutation (is it autosomal or sex-linked, recessive or dominant?) without knowing the location of the gene.
  • By observing the gene segregation (which alleles are passed on to which child) you can narrow down the location of a mutation to a certain locus - linkage interval.
9
Q

What is used to detect the presence of linkage?

A

A statistical test called the LOD score.

LOD score >3 is consistent with linkage.

LOD score

10
Q

What happens when you have determined a linkage interval?

A
  • Identify the genes within the interval.
  • Assess the genes as potential candidates based on their biological function.
  • Sequence the genes in the affected individuals to try and identify the causative mutation.
11
Q

What can confound linkage analysis?

A
  • Non-penetrance

- Phenocopies

12
Q

How can linkage analysis overcome the issue of genetic heterogeneity?

A

By performing linkage analysis in multiple pedigrees (families) and combining the results.

13
Q

Why is coding the human exome (all protein-coding regions) a cost-effective strategy for monogenic diseases?

A

The causative mutations in monogenic diseases have consistently been identified in the protein coding regions of the genome, so no need to sequence the entire thing at a higher cost.

14
Q

What do you gain from sequencing the exome?

A

You are able to catalogue all protein altering variations in an individual.

If the individual has a monogenic disease you can filter through the variation to identify which variant is causing the disease.

15
Q

If a person has a rare, autosomal dominant disease how would you filter through the variants in protein coding regions after sequencing their exome?

A

1) Because the disease is dominant, you ignore the homozygous variants and look at the heterozygous.
2) You look at the heterozygous mutations and see which ones are predicted to alter protein structure and which are silent.
3) Out of the variants predicted to alter protein structure, you ignore the variants that have been previously observed which leaves you which a number of novel candidate variants.
4) You then investigate these variants and dismiss the ones that do not match the phenotype of the disease.
5) For more accuracy, you compare the candidate variants of multiple unrelated individuals with the same disease.

16
Q

What is a de novo mutation?

A

Another term for a spontaneous mutation - when the mutation is not seen in the parents.

17
Q

What is a major confounding factor in exome sequencing for disease?

A

Genetic heterogeneity.

18
Q

What is a DNA variant/variation?

A

Where specific loci within the genome contain two or more alleles.

19
Q

What are transitions and transversions?

A

Transition = another word for a silent point mutation.

Transversion = a point mutation that changes the base chemistry = another word for missense or nonsense mutations.

20
Q

What do small rearrangements refer to?

A

Any changes in the DNA sequence whose effects are confined to a single exon of a gene.

21
Q

What are the consequences of variants in splice sites?

A
  • Exon skipping
  • Use of cryptic splice sites/new splice sites
  • Intron retention (small introns only)
  • Combination of above
22
Q

In which site are variants in DNA less commonly found?

A
  • Promoters
  • Untranslated regions
  • Polyadenylation signals
23
Q

What are amorphs and antimorphs?

A

Amorph = a variant that causes complete loss of gene function.

Antimorph = dominant alleles that act in opposition to normal gene activity.

24
Q

What are hypomorphs and hypermorphs?

A

Hypomorph = a variant that causes a partial loss of gene function.

Hypermorph = a variant that causes an increase in normal gene activity.

25
Q

What is a neomorphic mutation?

A

A variant that causes a dominant gain of gene function that is different from normal function.

26
Q

What changes to proteins causes loss of function phenotypes?

A
  • Little or no protein produced.
  • Protein is unstable or inappropriately targeted for degradation.
  • Residue or domain essential for protein function is missing or altered.
27
Q

How can loss of function alleles (which are usually recessive) display dominant inheritance?

A

1) Haploinsufficiency = the organism is so sensitive to protein levels that 50% reduction causes a noticeable phenotype.
2) Dominant negative effect = the mutated proteins disrupts the function of normal proteins in complexes.
3) Somatic second hits = the person inherits a defect and then has that defect amplified by another somatic mutation that affects the same protein.

28
Q

What are gain of function mutations?

A

Where rather than lose its principal function, the protein may become less specific in its normal function or acquire a novel function. These are dominantly inherited.

29
Q

What was the effected gene in infantile onset epilepsy in the Amish?

A

SIAT9 encoding the enzyme GM3 synthase which is used for the production of gangliosides which are vital for membrane stability. The mutation led to loss of function in this enzyme.

30
Q

Who is the consultand in a pedigree?

A

The person who initially approached the doctors with concerns over the condition in their family.

31
Q

What is genomic anticipation?

A

When the presentation of the disease gets worse as it is transmitted across generations. Seen in myotonic dystrophy and Huntington’s disease.

32
Q

What is genetic penetrance?

A

The proportion of carriers who manifest phenotypic symptoms of the condition - not all individuals who inherit a dominant mutation will necessarily show signs of the condition.

Penetrance is a statistic and is usually expressed as a percentage.

33
Q

What factor can increase the likelihood of a de novo mutation?

A

Paternal age due to the large number of mitotic divisions of male games stem cells during a man’s lifetime.

34
Q

What is germline mosaicism?

A

When a parent carries a small proportion of games that harbour the same mutation.

35
Q

What is compound heterozygosity?

A

Where someone has different allelic mutation at the same locus.

36
Q

What are lines of Blaschko?

A

Lines that represent territories of clonal cell populations on the skin.

37
Q

What are APO-E alleles?

A

They are alleles associated with Alzheimer’s disease.

  • e4 allele increases the risk of AD
  • e3 allele causes a baseline risk
  • e2 allele protects against AD
38
Q

What are genome-wide association studies?

A

They are tests to see whether the presence of a specific gene variant (SNP) increases the risk of disease. This is done across many individuals and by comparing affected and unaffected individuals.

39
Q

What diseases have absolute and relative risk?

A

Absolute = Mendelian diseases.

Relative = polygenic diseases.

40
Q

What P-value is used in GWAS? Why is it not 0.05?

A

5 x 10^-8

Because GWAS uses tens of thousands of people and the chance that any SNP has a causal effect on the phenotype being studied is very low.

41
Q

What is one of the most important aspects of genetic counselling?

A

To provide a risk figure for the patient, their offspring and other family members.

42
Q

Describe the process of next generation sequencing.

A
  • DNA is broken into smaller pieces by a sonar pulse.
  • DNA fragments are denatured (split into two strands) and hybridised to a surface (bead or solid plate).
  • Denatured DNA fragment is amplified in either an emulsion, PCR reaction or an isothermal bridge amplification reaction. Result is a clonal cluster of DNA.
  • The DNA is then sequenced together with fluorescent nucleotides (denatured parental strand has fluorescent nucleotides added).
43
Q

How do you calculate the probability of a child being affected by a genetic disease?

A

You multiply the probability of the child inheriting the disease by the penetrance of the disease.

44
Q

What is an obligate carrier?

A

An individual who may be clinically unaffected but who must carry a gene mutation based on analysis of the family history.

45
Q

What are most diseases and human traits caused by?/

A

Many genetic variants each having a tiny effect.

46
Q

What are genetic association studies?

A

They test the association between the alleles/genotypes of a SNP and the trait of interest.

47
Q

What are inheritance models?

A

The pattern of effects that different genotypes have on a phenotype is shown in these models.

On the Y axis you have the phenotype and on the X you have the genotype (AA, AB, BB).

48
Q

How are GWAS results usually displayed?

A

Manhattan Plot

X axis = association results across ordered chromosomes.

Y axis = P-values to log base 10.

49
Q

What is the key idea underlying the GWAS design?

A

That disease risk alleles pass from generation to generation together with other alleles that are nearby on the genome. Therefore, even if the variants are not identified in the study, the SNPs that are might be nearby and correlated with the variants. This is indirect association.

50
Q

What is a polygenic risk score?

A

They are constructed from the Manhattan plots created by GWAS data and predict an individual’s risk of disease based on the combination of their genotype and the effect size estimates from GWAS results.

51
Q

How to you calculate a polygenic risk score?

A

You put a horizontal line marking the P-value of 5x10^-8 on a Manhattan plot and take each variant that is above this line and put it into a score.

Often you take a whole series of scores using different P-value thresholds.

It is a sum of risk alleles from the individual’s genome-wide SNPs weighted by their GWAS-derived effect score (from the Manhattan plot).

This result is then compared to the disease state of the patient to see how accurate the score was (shown in a regression with a variety of P-value thresholds).

52
Q

How are polygenic risk scores useful for medical research?

A
  • Can assess shared genetic aetiology among phenotypes.
  • Act as a biomarker for disease.
  • Infer whether a biological factor is causally associated with a disorder.
  • Screen subjects for clinical trials.
53
Q

What is the future of polygenic risk scores?

A

Individuals can be placed on a spectrum of genetic burden to a disease/trait.

54
Q

What are the advantages of sequencing the whole genome?

A

A person’s genome doesn’t change so there is the potential to collect once, store and then refer to repeatedly for clinical care.

55
Q

What are the limitations of next generation sequencing?

A
  • Short reads of NGS (only small fragments are duplicated) make accurate characterisation of large variants hard.
  • NGS accuracy is currently lower than older, more expensive sequencing technology.
56
Q

What can you do to identify causal variants in the genome?

A
  • Filter variants that are frequently observed.
  • Look for variants identified as pathogenic.
  • Look for variants in genes linked to a condition.
  • Look for variants that effect functional elements.
  • Look for variants that are normally conserved across species.
57
Q

What is common among pathogenic variants?

A

A large proportion are false positives.

58
Q

What is rare disease diagnostics?

A

Where you sequence the affected individual and other family members (affected or unaffected).

59
Q

Who is whole-genome sequencing restricted to in clinical care?

A
  • People with monogenic diseases as they only have one variant.
  • People with clear phenotypes - can focus on the genes associated with the phenotype.
  • Patients who are ill.
60
Q

What is reported in clinical use of whole-genome sequencing?

A

Protein coding sequences only as it is easier to predict the effect of mutation.

61
Q

What were the objectives of the 100K genome project?

A
  • Improve health of NHS patients.
  • Stimulate wealth generation (economy).
  • Create legacy of infrastructure, human capacity and capability.
  • Enable large scale genomics research.
62
Q

What are the responsibilities of NHS genomic medicine centres?

A
  • Identifying and recruiting participants.

- Clinical care following results.

63
Q

What is human phenotype ontology?

A

A system that provides a standardised vocabulary of phenotypic abnormalities encountered in human disease.

64
Q

What information is fed back to the NHS from the 100k genome project?

A
  • Information about the patient’s main condition.
  • Information about additional ‘serious and actionable’ conditions (optional).
  • Carrier status for non-affected parents of children with rare diseases (optional).
65
Q

What are the three tiers of reported variants?

A

1) Variant is in the gene panel (in a list of genes known to be associated with a condition), there is clear loss-of-function evidence, and is a known pathogenic variant.
2) Variant is in the gene panel and is a missense mutation or a VUS (variant of unknown significance).
3) Gene is not in the panel (not in the known list of genes associated with a disease).

66
Q

Why has there been difficulties surrounding cancer in the 100K genome project?

A

Standard practice is to preserve tumours in formalin then fix them in paraffin for imaging, but this process causes damaged and broken DNA to be extracted for sequencing.

67
Q

What has been done to overcome the issues with tumour samples in the 100K genome project?

A

Tissue is now being frozen and effort is being made to ensure the sample is mainly tumour cells.

68
Q

What proteins do mitochondrial DNA code for?

A

Proteins involved in oxidative phosphorylation. All other proteins involved in repair of machinery and mitochondrial transcription and translation are nuclear proteins.

69
Q

What are the origins of mitochondrial diseases?

A

Both nuclear and mitochondrial DNA because they are so intimately linked.

70
Q

What are the general features of mitochondrial disorders?

A
  • Respiratory chain deficiency.
  • Reduction in cellular O2 consumption and ATP synthesis.
  • Increased lactic acid build-up in blood and CSF.
  • Overproduction of ROS instead of a reduction in ATP synthesis (sometimes).
  • May manifest in multiple systems and tissues.
71
Q

Which tissues are the most affected by mitochondrial disorders?

A

Those with high energy demand and are frequently involved in the neuromuscular system.

72
Q

What are the features of mitochondrial diseases caused by nuclear genome mutations?

A
  • Follow the same inheritance pattern as Mendelian disorders.
  • Can be caused by defects to genes involved in replication, transcription, translation and repair of MT DNA.

An example is Friedreich Ataxia

73
Q

What are the features of mitochondrial diseases caused by nuclear genome mutations with mitochondria dysfunction dependence?

A
  • Defects appear in genes that monitor and regulate mitochondria.
  • Requires mitochondrial dysfunction.
  • Important in ageing.

An example is Parkinson’s disease.

74
Q

What are the two genes affected in Parkinson’s disease?

A

Parkin

PINK1

75
Q

How does mitochondrial dysfunction cause Parkinson’s disease?

A

Normally, PINK1 is imported to the mitochondria, cleaved and released.

Damage to the mitochondria causes depolarisation of membranes leading to PINK1 accumulating in the outer membrane.

Parkin then recruited to the MT and tags it for autophagy.

76
Q

What are the features of mitochondrial diseases caused by mitochondrial genome mutations?

A
  • Caused by point mutations or re-arrangements.
  • Similar general features to disorders caused by nuclear mutations.
  • Inherited from the mother.
  • > 500 sites are linked to disease with the highest percentage (over 50%) in tRNAs.
77
Q

What is the most common mitochondrial disorder?

A

Leber’s hereditary optic nephropathy (LHON).

78
Q

What is medium-level heteroplasmy?

A

Where there are a mix of mutated and WT mitochondrial plasmids in a cell which is enough to cause mild symptoms of a mitochondrial disease.

79
Q

What is high level heteroplasmy?

A

Where there are far more mutated MT plasmids than WT leading to fatal encephalopathy (brain disease).

80
Q

What is the relation between complex diseases and mitochondrial diseases?

A
  • Small number of associations.
  • Causes susceptibility to complex diseases.
  • Most links are correlative, not validated and difficult to prove functionally.
81
Q

What is the link between mitochondria and cancer?

A

Some research has shown that in cancer you see an increased number of both homoplasmic and heteroplasmic mutations in tumour cells vs. normal cells.

However, there is very little proof that these mutations are linked to MT function or involved in tumourgenesis.

82
Q

What is euchromatin?

A

Lightly packed form of chromatin (DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription.

83
Q

Where is heterochromatin found in the cell?

A

On the inner membrane in contact with the peripheral nuclear lamina.

84
Q

What do epigenetic modifications influence and what does this regulate?

A

They influence how DNA is packaged into chromosomes and this regulates genome function.

85
Q

Where is epigenetic information reprogrammed?

A

On a genome wide level in the germ cells and early embryos.

86
Q

What puts methyl groups on DNA?

A

Methyltransferases

87
Q

What has occurred in the N-terminal tails of histones?

A

They have been post-transationally modified. Modifications are associated with active/silent genes, promoters, enhancers etc.

88
Q

What can the histone code be used for?

A

To predict regulatory regions.

89
Q

How do you identify non-traditional disease-causing mutations?

A

By identifying long-range enhancer mutations and other regions from GWAS.

Here we are linking diseases associations with regulatory information.

90
Q

What is genomic imprinting?

A

Where epigenetic changes persist (imprinted) through the germline and are passed on to offspring. However, these are reversible and open to re-setting in the next generation.

91
Q

What diseases are passed on as a result of genomic imprinting?

A
  • Angelman syndrome
  • Prader-Willi syndrome
  • Male infertility
  • Beckwith-Wiedeman syndrome
  • Silver-Russell syndrome
  • Albright heretidary osteodystrophy
  • Transient neonatal diabetes
92
Q

How many imprinted genes have been found in mice and humans?

A
  • 150 in mice

- 100 in humans

93
Q

Why are imprinted genes mis-expressed in uniparental disomies ( 2 copies of a chromosome come from the same parent)?

A

Because imprinted genes are expressed from only one parent.

94
Q

What are three types of epigenetic modifications?

A
  • Compaction/accessibility of DNA in chromatin.
  • Modification of core histones at the N-termini.
  • Methylation of DNA at CG dinucleotides.
95
Q

What genes are damaged in some cancers?

A

The genes that control cell growth.

96
Q

What happens when tumour suppressor genes are inactivated?

A

Allows cells to divide faster.

97
Q

What happens when oncogenes are activated?

A

Drives cells to divide faster.

98
Q

What are fusion genes?

A

Arise as a result of chromosome rearrangements. They typically give cells a growth advantage.

99
Q

What is multi-step carcinogenesis?

A

Where multiple genes are affected in the different stages of cancer development.

100
Q

Aside from tumour suppressor genes and oncogenes, what other gene is associated with cancer predisposition?

A

Damage/inactivation of DNA damage-response/repair genes. Cancer arises as a result of accumulation of mutations across the genome.

101
Q

What are three DNA repair mechanisms?

A

1) Mismatch repair.
2) Double-strand break repair.
3) Nucleotide excision repair.

102
Q

How are most cancer susceptibility genes passed on?

A

They are dominant with an incomplete penetrance causing them to appear to skip generations.

103
Q

What are the main features of cancer susceptibility genes?

A
  • Young age onset of cancer
  • Multiple primary cancers in the same person
  • Same type of cancer in several relatives
  • Recognisable pattern of cancers in family
104
Q

What is lynch syndrome?

A

A type of inherited cancer syndrome associated with a genetic predisposition to different cancer types. It arises from a mismatch repair deficiency.

105
Q

What is the Amsterdam criteria?

A

A set of diagnostic criteria used by doctors to help identify families which are likely to have lynch syndrome. The Bethesda criteria is used to trigger further investigation.

106
Q

How is lynch syndrome managed?

A
  • Daily low-dose aspirin
  • Regular colonoscopies from 25 years old
  • Discussion of risk-reducing surgery from 45 years old
107
Q

What is the lifetime cancer risk of a woman with the cancer susceptibility gene BRCA1?

A

Breast cancer = up to 80%

Ovarian = up too 50%

108
Q

What is Cowden syndrome?

A

An autosomal dominant inherited condition characterised by benign overgrowths called hamartomas as well as an increased lifetime risk of breast, thyroid, uterine, and other cancers. It is caused by germline PTEN mutations.

109
Q

What is Peutz-Jeghers syndrome?

A

A gastrointestinal polyposis disorder characterised by pigmented lesions on the lips and buccal mucosa. It causes increased risk of breast, GI and gynaecological tumours. It is caused by a germline mutation in STK11.

110
Q

What is Li-Fraumeni syndrome?

A

Condition also known as the sarcoma, breast, leukaemia and adrenal gland (SBLA) syndrome. Caused by a germline mutation in TP53. Radiation may induce cancer in this case.

111
Q

What is stratified medicine?

A

Based on identifying subgroups of patients with distinctive mechanisms of disease or particular response to treatments. This allows us to identify and develop treatments that are effective for particular groups of patients.

112
Q

What is the study of chromosomes called?

A

Cytogenetics

113
Q

What are the three types of chromosome abnormality?

A

1) Chromosome rearrangements
2) Whole chromosome aneuploidy
3) Copy number imbalance

114
Q

What is the cause of whole chromosome aneuploidy?

A

Non-disjunction at mitosis or meiosis.

115
Q

What is Robertsonian translocations?

A

They result from the fusion of two acrocentric (centromere is located close to the end of the chromosome) chromosomes = 13, 14, 15, 21, 22.

The most common are der(13;14) and der(14;21).

Balanced carriers are phenotypically normal but have reproductive risks.

116
Q

What are reciprocal translocations?

A

They can be between any segments of any non-homologous chromosomes. They are almost always unique in the family. Balanced carriers have reproductive risks that are dependent on the size of the translocation segments.

117
Q

What is pericentric inversion?

A

Where the inversion includes the centromere and there is a break point in each arm.

118
Q

What is a paracentric inversion?

A

Where the inversion does not include the centromere and both breaks occur in one arm of the chromosome.

119
Q

What are the traditional tests for prenatal cytogenetics?

A

G-banded chromosomes.

120
Q

What do you do if you need fast results for prenatal cytogenetics?

A
  • FISH

- QR-PCR

121
Q

What is a disadvantage of G-banded chromosome tests?

A

They have a resolution of ~5-10Mbs. This is a problem because many small imbalances (microdeletions) cause syndromic disease.

122
Q

What are examples of microdeletion syndromes?

A
  • DiGeorge
  • Williams
  • Angelman
  • Prader-Willi
  • Wolf-Hirshchhorn
123
Q

Why are submicroscopic chromosomal deletions recognised as syndromes?

A
  • Because they recur at low frequency in all populations.
  • Due to genomic structure at the disease locus this predisposes the patient to gene deletion-duplication by unequal recombination.
124
Q

What can array CGH (comparative genomic hybridisation) detect?

A
  • Whole chromosome aneuploidy
  • Microdeletion/duplication syndromes
  • Subtelomere imbalance
  • Other regions of imbalance (copy number variants etc.)
125
Q

What have research studies using array CGH shows?

A

That normal individuals carry multiple small CNVs (copy number variations). Different combinations of these CNVs may contribute to phenotypic variation between individuals.

126
Q

How does size of CNVs relate to inheritance?

A

The smaller the CNV, the more likely you are to inherit it.

127
Q

What can balanced translocations lead to?

A

Carrier of a balanced translocation can pass the rearrangement onto their offspring in an unbalanced form which is known as unbalanced translocation. Unbalanced rearrangements almost always give rise to a phenotype, the severity of which depends on the size of the rearrangement and chromosomes involved.

128
Q

What are the clinical features of psoriasis?

A
  • Itchiness
  • Cracked skin
  • Pain
  • Pitted nails
  • Arthritis (10-20%)
  • Social isolation
129
Q

What is the most and second most common type of psoriasis?

A

Most = plaque psoriasis (dry scaling patches)

Second = guttate psoriasis (drop-like dots occurring after strep or viral infection)

130
Q

What can inflammatory bowl disease be divided into?

A

1) Ulcerative colitis

2) Crohn’s disease

131
Q

What is ulcerative colitis?

A

Inflammation/ulcers only in the mucosa of the colon.

132
Q

What are the differences between ulcerative colitis and Crohn’s disease?

A

Ulcerative = continuous inflammation, only colon, superficial inflammation.

Crohn’s = patchy inflammation, mouth to anus involvement, full-thickness inflammation.

Both carry a risk of cancer and extra-intestinal manifestations.

133
Q

What are the four aetiological hypotheses for IBD?

A
  • Persistent infection
  • Defective mucosal integrity
  • Dysbiosis (decrease in protective bacteria and increase in aggressive opportunistic bacteria)
  • Dysregulated immune response
134
Q

What is pharmacogenetics?

A

The study of inherited genetic differences in drug metabolic pathways which can affect the individual’s responses to drugs.

135
Q

What do pharmacogenetics focus on?

A

Drugs with serious toxicity or a clear therapeutic benefit in optimising therapy.

136
Q

What is the CYP3A subfamily and what are they used for?

A

They are a subfamily of the most important drug-metabolising enzymes in the liver and the gut.

They are used as an immunosuppressant target in transplantation e.g. tacrolimus.

137
Q

Has CYP3A5*3 and tacrolimus entered routine clinical practice?

A

No

138
Q

What explains dose variation in Warfarin?

A
  • VKORC1 (polymorphisms)

- CYP2C9 (poor metabolites)

139
Q

How did pharmacogenetics affect warfarin?

A
  • Genotype-guided dosing of warfarin did not improve anticoagulation control.
  • Pharmacogenetic-based dosing was associated with a higher percentage of time in the therapeutic INR (international normalised ratio) range than standard dosing.
140
Q

What is allopurinol used for?

A

Treatment of gout.

141
Q

How are the pharmacogenetic adverse reactions of allopurinol avoided?

A

By avoiding using it and prescribing febuxostat instead - testing has not been done.

142
Q

What makes a good pharmacogenetic test?

A
  • Replication
  • Marker must have high effect/penetrance
  • Predictive but not prognostic
  • Clear patient benefit by reducing toxicity or improving efficacy
  • Testing should not delay therapy
  • Testing must be cost-effective
143
Q

What is azathioprine used for?

A

To treat IBD. It is the standard 1st line immunosuppression in IBD.

144
Q

What are the steps of treatment in Crohn’s disease?

A

1) Antibiotics
2) Corticosteroids
3) Azathioprine
4) Methotrexate
5) Biologics
6) Surgery

145
Q

What does thiopurine methyltransferase do to azathioprine?

A

Transforms it into thioguanine nucleotides which are cytotoxic.

146
Q

How are the potential cytotoxic effects of azathioprine avoided?

A

Patients are tested prior to starting therapy and the dose is adjusted depending on their TPMT results. This was deemed clinically useful and cost-effective.

147
Q

What are fluoropyrimidine drugs used for?

A

First line treatment for solid tumours including colorectal and breast cancer. They are antimetabolites.

148
Q

What will 10-20% of patients taking fluoropyrimidine drugs develop?

A

Severe (CTCAE grade >3) toxicity.

149
Q

What is the cause of fluoropyrimidine drug cytoxicity?

A

DPD (dihydropyrimidine dehydrogenase) deficiency

150
Q

Where can DPD enzymes not be assayed?

A

Red blood cells

151
Q

How is fluoropyrimidine toxicity avoided in the UK?

A

DPYD genotyping prior to starting fluoropyrimidine therapy is done by a number of hospitals in the UK and Ireland. However, each case requires a business case for testing as it is not very cost effective.

152
Q

What is gene therapy?

A

The use of recombinant genetic material (DNA, RNA or hybrid molecules) under different forms or pharmaceutical preparations as a therapeutic agent.

153
Q

What are the three components of a gene therapy protocol?

A

1) Target tissue
2) Efficient gene delivery
3) Transcriptional control

154
Q

What is the ideal cell target for gene therapy?

A

Pluripotent, self-regenerating stem cells from the patient being tested.

155
Q

What are examples of non-integrating viral gene therapy vectors?

A
  • Adenovirus

- Adeno-associated virus

156
Q

What are examples of integrating viral gene therapy vectors?

A
  • Retrovirus

- Lentivirus

157
Q

What level of regulation of the transcription unit do you need for acquired diseases (such as HIV) and inherited diseases?

A

Acquired = transient and long term.

Inherited = long-term only.

158
Q

Which diseases have shown successes via in vivo gene therapy procedures?

A
  • Haemophilia B
  • Parkinson’s disease
  • Leber’s congenital amaurosis

All of these have had an AAV vector delivered directly into the desired tissue.

159
Q

What is the ex-vivo gene delivery protocol?

A
  • Cells are harvested under general anaesthesia and stem cells are isolated.
  • Cells are activated to grow for 24 hours with cytokines.
  • Cells are then transduced by three rounds of culture in the presence of the supernatant containing the retroviral vector in sterile bags.
  • The transduced cells are infused back into the patient.
160
Q

What were the severe adverse effects of the clinical trials for SCID-X1? What does this mean?

A

6 patients developed T-lymphocyte proliferative disease.

Means there is a clear need for improved vector design with reduced insertional mutagenesis potential.

161
Q

What is SCID-ADA?

A

A deficiency of adenosine deaminase (ADA). This is a purine metabolic defefct that leads to accumulation of toxic metabolites leading primarily to impaired lymphoctye development and function.

162
Q

What is Strimvelis?

A

The second approved gene medicine in the EU.

163
Q

What is Wiskott-Aldrich Syndrome?

A

An X-linked mutation in the WAS gene that encodes for the WASP protein that regulates the cytoskeleton.

It leads to infections, microthrombocytopenia, eczema, autoimmunity, and
lymphoid malignancies.

164
Q

What form of gene therapy is used to treat patients with Wiskott-Aldrich syndrome?

A

Lentiviral haematopoietic stem cell gene therapy.

All patients showed improvement.

165
Q

What is X-CGD?

A

Also known as granulomatous disease it is a primary immunodeficiency caused by a defect in the oxidative anitmicrobial activity of phagocytes (specifically neutrophils). Mutations are found in the NADPH oxidase complex.

70% of cases result from defects in a X-linked gene.

166
Q

Did gene therapy for X-CGD work?

A

Showed restoration of anti-microbial activity in gene-corrected cells.

However, one patient died of severe bacterial sepsis after colon perforation 27 months after the therapy.

167
Q

Why did the X-CGD gene therapy trial need transcriptional control elements?

A

They would be able to negate insertion site position defects, avoid epigenetic mediated transgene silencing and provide stable, reproducible levels of therapeutic gene expression.

168
Q

What is junctional epidermolysis bullosa?

A

Mutations in genes encoding for basement membrane component laminin 5 causes devastating and often fatal skin adhesion disorder.

169
Q

What was the result for the gene therapy for junctional epidermolysis bullosa?

A

Complete epidermal regeneration and normal-looking epidermis was maintained throughout the 1 year follow-up. Full, normal and robust skin function was restored.

170
Q

What was the first use of a lentiviral vector for inherited disorders? What was the result?

A

Gene therapy for X-linked adrenoleukodystrophy (ALD).

Progressive cerebral demyelination stopped. Results were comparable to stem cell transplantation. Only one patient was not helped.

171
Q

What is the gene therapy required to treat β-haemoglobinopathies?

A

Requires stable introduction of normal functioning β-globin transcriptional uni into the haematopoietic stem cells of the patient. A minimum of 25% normal levels of β-globin is required for a therapeutic effect and 50% for a cure.

Lentiviral vectors were used.

172
Q

What are the future advances to make gene therapy a widely applicable technology?

A
  • In vivo targeted gene delivery systems
  • Efficient non-viral vectors
  • Minimise the risks such as insertional mutagenesis
  • Gene editing where applicable