Genome sequencing Flashcards
(20 cards)
What is the significance of the completion of the Human Genome Project?
The completion of the Human Genome Project provided a complete blueprint of human DNA, revolutionizing human biology by enabling the identification of disease genes, development of gene therapies, and a deeper understanding of human health and disease.
Explain the concept of ‘positional cloning’ and why it was a challenging technique in human genetics.
Positional cloning was a laborious and time-consuming technique used to identify genes based on their location on chromosomes. It involved using genetic markers and linkage analysis to narrow down the region containing the gene of interest, making it a challenging process before the availability of the human genome sequence.
Why is it crucial to sequence the DNA of both parents and the affected child when investigating a de novo mutation?
Sequencing the DNA of both parents and the affected child allows researchers to identify mutations specific to the child. By comparing the child’s DNA to the parents’, variants inherited from the parents can be ruled out, increasing the chances of finding the disease-causing de novo mutation.
What are the key characteristics of rare diseases, and how has genome sequencing impacted their understanding and diagnosis?
Rare diseases are individually uncommon, affecting less than 1 in 2000 people, but collectively affect a significant portion of the population. Genome sequencing has revolutionized their diagnosis by enabling the identification of causative genes, leading to a better understanding of these disorders and fostering connections between affected families worldwide.
Describe the mechanism of action of Nusinersen in treating Spinal Muscular Atrophy (SMA).
Nusinersen is an oligonucleotide drug that targets the SMN2 gene, promoting the inclusion of exon 7 during splicing. This results in increased production of functional SMN protein, which partially compensates for the deficiency caused by mutations in the SMN1 gene in SMA patients.
Explain how the understanding of hereditary persistence of foetal haemoglobin (HPFH) has led to therapeutic strategies for sickle cell disease.
Individuals with HPFH continue to produce foetal haemoglobin throughout their lives. Clinical observations revealed that sickle cell disease patients with co-existing HPFH experienced milder symptoms, indicating that foetal haemoglobin could compensate for the defective adult haemoglobin. This led to therapeutic strategies targeting the reactivation of foetal haemoglobin production in sickle cell disease.
How has transcriptome sequencing, particularly single-cell sequencing, advanced our understanding of diseases like medulloblastoma?
Transcriptome sequencing, specifically single-cell sequencing, has allowed researchers to analyze gene expression profiles of individual cells within a tumor. In medulloblastoma, this revealed four distinct subtypes with different underlying genetic causes, providing valuable insights into tumor heterogeneity and potential therapeutic targets.
What are Genome-Wide Association Studies (GWAS), and what are their limitations in understanding complex genetic disorders?
GWAS are large-scale studies that compare genomes of individuals with and without a particular disease to identify genetic variations associated with the disease. However, they often identify many common variants with small effects, making it difficult to pinpoint specific causative genes and understand the complex interplay of genetic and environmental factors in these disorders.
Describe the role of biobanks in studying the interplay of genetics and environmental factors in complex diseases.
Biobanks are large-scale repositories of biological samples and health information from a diverse population. By combining genetic data, health records, and lifestyle information, they provide a powerful tool for studying the complex interplay of genes and environment in the development of diseases like heart disease, diabetes, and cancer.
How has genome sequencing contributed to our understanding of infectious diseases, specifically focusing on COVID-19?
Genome sequencing of the SARS-CoV-2 virus allowed for rapid development of diagnostic tests and vaccines. Additionally, GWAS on COVID-19 patients identified genetic variations associated with disease severity, providing insights into the host factors influencing susceptibility and immune responses to the infection.
How does genome sequencing help combat drug resistance?
Enables rapid molecular diagnostics, which is key for early detection which guides therapy
Whole-genome sequencing enables precise mapping of resistance mutations and transmission chains in drug resistant diseases like TB
What is DM-seq and why is it better than other sequencing methods?
DM-seq (Damage-seq or DNA Modification sequencing)
Direct Methylation Sequencing (DM-seq) nondestructively maps 5mCs directly at single-base resolution with enzymes, and only needs nanograms of sample
Designed to map DNA damage or DNA modifications (like methylation).
Can be personalised: A damage-specific antibody or chemical treatment may be used to enrich for damaged DNA, followed by adapter ligation and sequencing.
Way less destructive than BS-seq and more resolution than other damage assays (ELISA)
5mC is the normal cytosine nucleotide in DNA that has been modified by the addition of a methyl group to its 5th carbon. The role of this mark is so distinct that many consider 5mC to be the “5th base” of DNA.
5mC is a very important repressor of transcription in the genome. When present in promoters, 5mC is associated with stable, long-term transcriptional silencing. This may occur by either blocking positive transcription factors, or promoting the binding of negative ones
What gap in technology/knowledge did Next Gen sequencing fill?
1) Sanger sequencing could only process one DNA fragment at a time so sequencing whole genomes was slow and labor-intensive
2) Expensive
3) Sangar was limited to small known sequences
4) Wasn’t able to be used in the clinic
What is bridge RNA and how does it address limitations of CRISPR?
Bridge RNA (bRNA)
bRNA is a synthetic or engineered RNA molecule that can
1) bring together bits of CRISPR (Cas effector protein and a guide RNA)
2) recruit different effectors to specific RNA/DNA (aka programmable targetting)
3) Reduces CRISPR off target effects and allows CRISPR to function in only specific contexts by requring dual recognition of both DNA sequence and a RNA signal
What contributions has single cell RNA sequencing made to understanding health and disease?
scRNA-seq has made it possible to zoom in on individual cells, revealing how diseases arise, progress, and respond to therapy
used in:
- Cancer diagnosis: Revealed intra-tumoral heterogeneity in glioblastoma and melanoma
- Personalised medicine: scRNA-seq of biopsy samples in leukemia or breast cancer can guide tailored treatment decisions
- Infectious disease: During COVID-19, scRNA-seq helped map how SARS-CoV-2 affects different lung and immune cells
- Regenerative medicine: used to make protocols for inducing iPSCs; tracks stem cell differentiation and identifies key regulators for tissue regeneration
What are the variations of genome sequencing?
- Whole genome sequencing
- Whole exome sequencing
- Transcriptome sequencing
- Single cell sequencing
Whats the imapct of genome sequencing?
- Identifying casual mutations
- Understanding rare disorders
- Long-term population studies (UK Biobank)
- Insights into tumors (allowing for personalised medicine)
- Revolutionised diagnosis, treatment, prevention (of NDs and cancer)
How is genome sequencing used in research for NDDs?
- exome sequencing in parent-child trios
- SHANK3 organoid models (understanding function/impairment)
- Loci-specific epigenome editing (dCas9-SunTag) (for FMR1 reactivation, or Mecp2 function)
identify genetic mutations and variations
deactivated Cas9 (dCas9) cannot cut DNA, but can recognize and bind to a specific location and recruit effector protein (like a demethylation protein), allowing high fidelity activity targetting
FXS: dCas9-SunTag was fused to Tet1 demethylase enzyme as a potential therapeutic, shown to be effective in iPSCs from FXS patients
Mecp2: Targetted methylation of Mecp2 in mice decreased expression and increased ASD-behaviours, useful for creating mouse model
How did NGS revolutionise diagnostics?
- Made DNA sequencing rapid and cheap; lung cancer diagnosis evolved from pathology based to gene based (overexpressed targets like EGF receptor)
- Genome sequencing for rare genetic diseases (3-4x increase in identifying medelian genes), eg NDD diagnosis
- Transcriptome sequencing; eg when combined with whole-genome sequencing of medullablastoma lead to personalised treatment approach for kids
- Proteomics like Reverse Phase Protein Microarray used to find pathway signatures in patient subgroups and link them to drug responses
- Rapid diagnostic tests for malaria are based off of whole-genome sequencing in Plasmodium falciparum
What is sickle cell disease?
- point mutation in the HBB gene (makes hemoglobin) causes it to clump and RBCs have a sickle shape
- Sickled cells can’t pass through small blood vessels bc of shape which is super painful (“sickling crises”)
- NGS and genome-seq made identifying mutations in the gene possible, which lead to targetted therapeutic development (eg increasing HbF via BCL11A gene therapy or edited-HSPC therapy, or AAV6+RNP therapy)
BCL11A is a transcriptional repressor that normally silences fetal γ-globin gene expression during the switch from fetal to adult hemoglobin. Inhibiting the enhancer of the BCLL1A gene inhibits this silencing, maintaining HbF and leveraging its protective ability
A research study detailed an ex vivo gene editing strategy involving patient-derived HSPCs, using CRISPR-Cas9 delivered as ribonucleoprotein (RNP) complexes in conjunction with adeno-associated virus serotype 6 (AAV6) vectors for donor template delivery (it was effective in the cells)
