genomics/personalised medicine Flashcards
two types of schizophrenia
Demhaja (2014) = treatment-resistant have normal 18F-DOPA uptake (PET) but elevated ACC glutamate (anterior cingulate cortex), patients that responded had higher 18F-DOPA uptake but normal ACC glutamate = evidence for 2 distinct subtypes of schizophrenia
- 2 hypothesis = dysregulation of dopamine and NMDA hypofunction = are not linked, 2 different subtypes
breast cancer classification
van’t Veer et al (2002)
• Background
- If we can identify BC early = usually responds to surgery
- amongst breast cancer patients, chemotherapy reduces risk of breast cancer metastasis by about 30%, but around 75% patients receive no clinical benefit
- since chemotherapy is highly unpleasant for the patient + can be dangerous, ideally it would only be given to patients who derive some clinical benefit
• Findings
- performed microarray analysis of 78 primary breast cancer tumours identified a gene expression ‘profile’ of 70 genes that predicts risk of future distant metastasis more accurately than any other measure = could identify in advance which patients did/didn’t need chemotherapy
o The group with a ‘good’ as opposed to a ‘poor’ prognosis did not suffer distant metastases within 5 years
- Computer software can now compare a patient’s mRNA profile to the gene expression profile from this study in order to predict whether they would benefit from chemotherapy
- FDA approved this test 5 years later = named the MammaPrint test, which helps the clinician to identify high and low risk patients and so make an informed decision and personalise the treatment of patients with early breast cancer
• Conclusion = powerful example of how a personalised medicine approach based on sub-classification of tumours has already made it into the clinic, allows subdivision of histologically similar tumours
• This is an example of how combined genomics and bioinformatics efforts can be applied in a clinical setting to make a real different to patient treatment
• Limitations = however, the clinical utility of this test does remain controversial
- Cardoso et al. (2016) published a phase III randomised study involving 6693 women with early-stage breast cancer, which found that only ~46% of patients considered to be a low genomic risk by the MammaPrint test but a high clinical risk (by looking at conventional histology) might not require chemotherapy. Thus more RCTs are required to validate its utility
tumour heterogeneity
Tumour heterogeneity = Gerlinger et al (2012)
• studied colorectal cancer (CRC) samples and their associated metastatic sites by performing whole-exome sequencing, chromosome aberration analysis and ploidy profiling
• Phylogenetic reconstitution revealed branched evolutionary tumour growth, with 63-69% of all somatic mutations not being detectable across every tumour region
• There was extensive heterogeneity within each tumour, with gene expression signatures of good and poor prognosis being detected in different regions of the same tumour
• This intratumour heterogeneity can cause us to underestimate the tumour genetic landscape from a single biopsy sample, which underlies the need to study biopsies from different regions of the tumour, and different metastases if we are to accurately predict therapeutic response, although this itself has practical limitations
HIV GWAS
- GWAS by the International HIV controllers study 2011 comparing HIV-1 controllers and progressors found over 300 SNPs within the MHC and none elsewhere. From this specific amino acid positions (67,70 and 97) within the HLA-B peptide binding grove were shown to have stronger associations with host control than any single classical HLA allele. This suggests conformational differences in peptide presentation at these sites and subsequent T cell functionality contribute to the protective phenotype of HIV controllers.
schizophrenia GWAS
- The Schizophrenia Working Group of the Psychiatric Genomics Consortium in 2014 published an analysis of almost 37,000 pts and 113,000 controls investigating SNPs
o They identified 108 loci which implicated around 600 genes with a statistically significant association to schizophrenia.
o This highlighted the importance of several established risk genes, such as DRD2 involved in dopaminergic singalling and GRIN2B, DISC1 and NRG1/ERBB4 involved in glutamatergic NMDA rececptor singalling
o 83 of these conservatively defined loci had not been implicated before, thus new associations were also found with CACNA1C and CACNB2, which code for voltage-gated calcium channel subunits
o Moreover, associations were also significantly enriched at enhancers active in cells with important immune functions, particularly B-lymphocytes (CD20 and CD19), suggesting that the immune system could somehow be involved in schizophrenia risk
RNAi discovery
Fire et al (1998) = C.elegans
- injected both sense and antisense mRNA of unc-22 gene (encoding myofilament) caused significant silencing of this gene, abolished protein expression and caused severe twitching phenotype
- discovered that presence of dsRNA caused silencing of specific gene by specific elimination of endogenous mRNA transcript
- tested hypothesis by injecting other dsRNAs for other worm proteins observed effective silencing of respective genes (including GFP in transgenic worm cell line which led to loss of fluorescence)
- C. elegans good for this study = small simple animal so easy to deliver dsRNA by injection, transparent so easy to visualise effects
screening for genes in rare genetic disorders
- Yamamoto et al. 2014 performed a mosaic genetic screen of lethal mutations on the Drosophila X chromosome and identified 165 genes necessary for development, maintenance and function of the nervous system. Simultaneously, they investigated rare variants in 1929 human exomes from families with unsolved Mendelian diseases
- They merged the human and fly datasets, and identified the ANKLE2 gene (which codes for a mitotic regulator), where in a recessive disorder, it was possible to show that ANKLE2 variant was responsible for a microcephaly phenotype, both in humans and flies.
- The impact of this work can go far beyond the impact on a specific family that has this rare disorder – Ankle2 is an example of that.
- Shah et al. 2018 subsequently showed that ANKLE2 protein interacts with a Zika protein called NS4A. By introducing this protein into Drosophila, it has been possible to show that this protein can also induce microcephaly this suggested that Ankle2 might be a critical protein in the developmental disorders that are associated with Zika infections
drosophila modelling
Moreover, we can now model cancers such as colorectal cancer (CRC) in Drosophila, so it is now possible to introduce all the relevant CRC mutations into the flies and then test drugs on the flies to see which are best at rescuing lethality. In this manner, Drosophila hold significant promise to be a powerful tool for personalised medicine in the field of oncology.
- One such example of their success was in a study by Bangi et al. (2019), who used Drosophila modelling to develop personalised treatment for a patient with treatment-resistant metastatic KRAS-mutant CRC.
- They first performed genomic analysis of the tumour, and identified 9 key driver mutations, which then modelled in Drosophila
- They then carried out a robotics-based screen which identified trametinib plus zoledronate as an optimal treatment combination
- Treating the patient with this combination led to a strong response, with the lesions remaining stable for 11 months.
Thus, a personalised treatment approach holds huge promise for identifying the best treatment option or combination in patients with treatment-resistant cancers.
ILCs in physiology
- Moro et al (2010) = ILC2s are essential in the immune response to the parasite Nippostrongylus brasiliensis
- observed that mesentery production of IL-5 and IL-13 and goblet cell hyperplasia was only seen in Rag2-/- mice infected with the parasite, and not Rag2-/- IL2Rg-/- mice indicated that ILC2s are an important source of these cytokines
- also showed that adoptive transfer of normal ICL2s into Rag2-/- IL2Rg-/- mice was sufficient to induce IL-13 production and GC hyperplasia
- CD4+ transfer did not induce IL-13 production until 2 weeks later
- Indicates that IL-13 in early infection must be ILC2-derived and that ILC2s likely play non-redundant roles in the early response to helminths
CD1 knockout
o Simple CD1 knockout mice were produced by Yu et al. (2001), which were crossed with ErbB2-MMTV transgenic mice (i.e. breast-cancer susceptible mice expressing the oncogene ErbB2 under a mouse mammary tumour virus promoter)
o These mice did not develop breast cancer, indicating CD1 was necessary for the oncogenic properties of ErbB2.
o However, since the CD1 mutation was introduced in the germline, this does not reflect normal development, so the knockout may interfere with normal developmental processes.
CD1 conditional KO
o Subsequently, Choi et al. (2012) investigated the role of CD1 at different stages in cancer development by producing tamoxifen-inducible CD1 conditional knockout mice
o They crossed these with ErbB2-MMTV mice and found that the induction of tamoxifen halted breast cancer development in the ErbB2-MMTV mice, and led to senescence of tumour cells. Thus, the development of breast cancer became ‘dependent’ on CD1
- This highlights the importance of not making assumptions from simple knockout of gene function without exploring conditional knockouts.
- There are however caveats to this experiment, for example their mice had increased ErbB2 in all cells, which is not the case in humans. And unlike genetic models, human cancers are heterogeneous and germline mutations are rare in human cancers – they are mostly are stochastic, and advanced tumours accumulate mutations over time. Thus tumour development in humans is likely to be less dependent on any given mutated gene than in experimental tumours
- But on the other hand, an advantage was that all the mice were genetically identical to controls (which could not be achieved in humans).
HIV SCID-hu mice
Gaudin et al, 1997: first study to evaluate protection using Ab infusion – gave 1 mg/kg of bn Ab b12 (very high dose!), and showed 100% protection after an IV (!) challenge = SCID-hu mice
optogenetics
light-activated channel under control of specific promoter in brain = shine light on mouse = activates that neuron population
• Pascoli (2015) – showed sufficiency of stimulating mesolimbic dopaminergic neurons for progression of addiction
o They produced DAP-Cre mice (where DAP = the dopamine transporter) and injected them with AAV expressing channelrhodopsin-2 (ChR2) and yellow fluorescent protein
o This produced specific expression of ChR2 in dopaminergic mesolimbic neurons
o They then placed the mice in a Skinner box in which the mice could press a lever, which elicited a burst of laser stimulation in the VTA
o This induced phasic firing in dopaminergic neurons, and led to a strong reinforcement of lever pressing
invasive (craniotomy) or scattered light, differs to endogenous firing pattern, conceals heterogeneity
ILCs in disease
whilst Buonocore (2013) showed the importance of ILC1s and ILC3s in colitis. In these experiments, they induced innate immune colitis in Rag-/- mice through infection with Helicobacter hepaticus and found that the number of ILCs in the intestine increased by 100-fold during infection, and that depletion of ILCs using Thy1 antibodies completely abrogated colitis and typhlitis. However, like many others, these studies indicate involvement but not non-redundancy; generation of good models lacking ILC1s and ILC3s in the presence of normal T cell populations is required to identify if ILCs are indeed essential. Not necessarily linked to the question = IBD inflammation is probably an unfortunate side effect rather than a role for ILCs in themselves
