2DEBBY HELLEBREKERS – CLINISCHE GENETICA
Inhoud
Workshop 1 – FISH............................................................................................................................... 10
Journal Club 1 – ROS1 gene fusions detection approaches in cancer ................................................... 12
Case 1 – When Omics meets Genetics .................................................................................................. 13
Chromosome Rearrangements....................................................................................................... 13
Chromosomal Copy Number Variants .......................................................................................... 13
1) What is Leigh Syndrome? What are some symptoms and what type of disease is it? What is the
cause? What are the metabolic consequences?.................................................................................. 13
Symptoms ...................................................................................................................................... 14
Genomic Causes ............................................................................................................................ 14
2) What is PGD? What techniques can be used? Why is it used? And what are the limitations of
PGD considering metabolic/polygenic diseases? .............................................................................. 14
Fish In Situ Hybridisation (FISH) ................................................................................................. 15
Comparative Genome Hybridisation (CGH) (NOT USED)...................................................... 15
Polymerase Chain Reaction (PCR) ............................................................................................... 15
Next Generation Sequencing (NGS) ............................................................................................. 15
Preimplantation Genetic Haplotyping (PGH)................................................................................ 16
3) What is usually used to determine Leigh Syndrome (metabolic disorders in general) in a
clinical setting? .................................................................................................................................. 16
General Diagnostics for IEMs ....................................................................................................... 16
4) What are the techniques used for transcriptomic analysis? And what is their added value?
(WES is the general golden standard, but cannot explain every problem on its own) - Brief
overview of metabolomics versus transcriptomics and their added value ........................................ 17
Micro-Arrays ................................................................................................................................. 18
RNA-Seq ....................................................................................................................................... 18
DNA Sequencing ........................................................................................................................... 19
Metabolomics ................................................................................................................................ 20
Transcriptomics ............................................................................................................................. 20
Proteomics ..................................................................................................................................... 21
5) What is the advantage of transcriptomics over WES combined with Metabolomics? .............. 21
Case 2 – Identical or Not? ..................................................................................................................... 22
1) What is ALS? How does it come about? How heritable is ALS? Multifactorial stuff
(environmental effects): Short summary (mainly genetic basis) ....................................................... 22
ALS and Symptoms....................................................................................................................... 22
Causes............................................................................................................................................ 22
Pathophysiology ............................................................................................................................ 22
1
, 2) How can monozygosity or dizygosity of twins be determined? And how can genetically
identical twins have different phenotypes? (Partial heterochromia example in the case) ................. 23
Physical Features ........................................................................................................................... 23
Placenta-based examination .......................................................................................................... 23
DNA-Testing ................................................................................................................................. 23
Random Interesting Info ................................................................................................................ 24
Sectoral Heterochromia ................................................................................................................. 24
3) Which tests are online available for genetic testing? What technologies do the companies use
(especially the Twin-Testing Site and 23andMe)? What information do those techniques provide?
How much does it cost? → Direct consumer testing for health and/or “fun”.................................... 24
Types of DTC Genetic Test ........................................................................................................... 25
EasyDNA....................................................................................................................................... 25
23andMe ........................................................................................................................................ 25
Illumina Chips for Genotyping SNPs ............................................................................................ 26
4) Look into the lecture for cases/examples of WGS of monozygotic twins? What were these
research results? ................................................................................................................................ 26
Differences in Monozygotic Twins ............................................................................................... 26
5) Ethical and Legal aspects .......................................................................................................... 28
Ethical Considerations ................................................................................................................... 28
Legal Aspects ................................................................................................................................ 28
Workshop 2 – WES Analysis ................................................................................................................ 29
Journal Club 2 – Prime Editing ............................................................................................................. 30
Case 3 – Pick the Model that Suits You ................................................................................................ 32
1) What is Autosomal Recessive Cerebellar Ataxia (ARCA)?...................................................... 32
Cerebellar Ataxia ........................................................................................................................... 32
Ataxia and Possible Molecular Causes.......................................................................................... 32
2) Which in silico methods are available for predicting the genetical testing? a) What are their
advantages and disadvantages? ......................................................................................................... 34
Databases ....................................................................................................................................... 34
Software Programs and their Utility .............................................................................................. 34
Advantages .................................................................................................................................... 34
Disadvantages................................................................................................................................ 35
3) Which in vitro methods are available for genetic testing? a) What are their advantages and
disadvantages? b) What are their ethical concerns for such methods? ........................................... 35
Traditional in vitro cell cultures .................................................................................................... 35
3D Cultures and Organoids ........................................................................................................... 35
2
, 4) What are conventional animal models used within genetical research? (EUROPEAN
LEGISLATION: WAD?) a) What are their advantages and disadvantages? b) What are the ethical
concerns for each of them? ................................................................................................................ 36
3Rs ................................................................................................................................................. 36
Invertebrates ONLY DROSOPHILA HAS A GENOME-WIDE KNOCKDOWN
LIBRARY!!!! 36
Vertebrates..................................................................................................................................... 37
Pure and Applied Research ........................................................................................................... 38
Ethical Considerations ................................................................................................................... 38
Alternatives to Animal Testing ..................................................................................................... 39
(ARCA) Considerations ................................................................................................................ 39
5) Propose an experimental set-up for investigating the genetical findings within the case? In
Silico, in vitro, in vivo, what models can be specifically used in this case, and why or why not? ... 40
In Vitro cell tissue Notes ............................................................................................................... 40
Case 4 – ................................................................................................................................................. 41
1) What is known and what is not known about the human genome? ........................................... 41
Completeness of the Human Genome Sequence ........................................................................... 41
Protein Coding Genes (PCGs) ....................................................................................................... 41
Non-Coding DNA ......................................................................................................................... 42
2) What are the advantages and disadvantages of Forward Genetics approaches? Pick an Example
(JC Paper): What is Genetic Screening? ........................................................................................... 43
Forward Genetic Screening ........................................................................................................... 43
Reverse Genetic Screening ............................................................................................................ 45
Mapping Mutants .......................................................................................................................... 46
Journal Club Genetic Death-Screening ......................................................................................... 46
Laboratory Mouse Breeding .......................................................................................................... 46
2021 ............................................................................................................................................... 47
3) What does it look like in an animal model? .............................................................................. 47
4) Apply the knowledge to the case, focussing on nDNA (rather than mtDNA) .......................... 48
Presentation Notes and JC ..................................................................................................................... 50
Case 5 – ................................................................................................................................................. 52
1) What are proto-oncogenes and tumour suppressor genes (TSGs)? What are general treatments
for gain-of-function and loss-of-function treatments for cancers? .................................................... 52
General Treatments ....................................................................................................................... 53
2) What are current principles/techniques/drugs used to treat gain-of-function mutant proto-
oncogenes? ........................................................................................................................................ 53
Oncogene Addiction ...................................................................................................................... 53
3) Why can loss-of-function genes not be properly treated for cancer? ........................................ 55
3
, 4) What is synthetic lethality? And can it be abused for drug treatments for certain cancers? i.e.
BRCA ................................................................................................................................................ 57
Biological Importance ................................................................................................................... 57
5) How can you screen for synthetic lethality processes in cancer? And thereby find novel drug-
targets? Look for a BRCA paper ....................................................................................................... 58
Screening ....................................................................................................................................... 58
6) How can cancers become resilient against the PARP-inhibitor treatment in case of BRCA-
cancers? ............................................................................................................................................. 59
BRCA’s Achilles Heel .................................................................................................................. 60
PARP Inhibitor Principle............................................................................................................... 60
PARP-Inhibitor Resistance............................................................................................................ 60
Case 6 – ................................................................................................................................................. 61
1) What is the Krebs-Cycle and how is it involved within regulating epigenetic modifications? . 61
NADPH ......................................................................................................................................... 61
Cancer............................................................................................................................................ 61
Epigenetics .................................................................................................................................... 61
2) What are the pathophysiology and pathogenesis of Glioblastoma? .......................................... 63
Pathogenesis .................................................................................................................................. 63
3) What do IDH-1/2 normally do? And what goes wrong within neuroblastoma? ....................... 64
Isocitrate Dehydrogenase-1/2 (IDH1/2) ........................................................................................ 64
IDH1/2 in Cancer .......................................................................................................................... 66
4) What does MGMT normally do? And what goes wrong within neuroblastoma? ..................... 68
MGMT........................................................................................................................................... 68
Temozolomide ............................................................................................................................... 68
5) What is the current standard care for glioblastoma? Can IDH-1/2 and/or MGMT be used as
biomarkers for Glioblastomas? Are they viable therapeutic targets? ................................................ 69
Current Standards .......................................................................................................................... 69
Biomarkers .................................................................................................................................... 69
Therapeutic Targets ....................................................................................................................... 70
Journal Club 4 and Project stuff – Mutatome – ..................................................................................... 72
Case 7 – Cardiogenetics ........................................................................................................................ 74
1) What is Cardiac Disease and sudden cardiac death? ................................................................. 74
Epidemiology ................................................................................................................................ 74
Pathophysiology ............................................................................................................................ 74
2) What are the physiological differences between a normal and athlete’s heart? ........................ 75
Signs and Symptoms ..................................................................................................................... 75
Cause ............................................................................................................................................. 75
4
Inhoud
Workshop 1 – FISH............................................................................................................................... 10
Journal Club 1 – ROS1 gene fusions detection approaches in cancer ................................................... 12
Case 1 – When Omics meets Genetics .................................................................................................. 13
Chromosome Rearrangements....................................................................................................... 13
Chromosomal Copy Number Variants .......................................................................................... 13
1) What is Leigh Syndrome? What are some symptoms and what type of disease is it? What is the
cause? What are the metabolic consequences?.................................................................................. 13
Symptoms ...................................................................................................................................... 14
Genomic Causes ............................................................................................................................ 14
2) What is PGD? What techniques can be used? Why is it used? And what are the limitations of
PGD considering metabolic/polygenic diseases? .............................................................................. 14
Fish In Situ Hybridisation (FISH) ................................................................................................. 15
Comparative Genome Hybridisation (CGH) (NOT USED)...................................................... 15
Polymerase Chain Reaction (PCR) ............................................................................................... 15
Next Generation Sequencing (NGS) ............................................................................................. 15
Preimplantation Genetic Haplotyping (PGH)................................................................................ 16
3) What is usually used to determine Leigh Syndrome (metabolic disorders in general) in a
clinical setting? .................................................................................................................................. 16
General Diagnostics for IEMs ....................................................................................................... 16
4) What are the techniques used for transcriptomic analysis? And what is their added value?
(WES is the general golden standard, but cannot explain every problem on its own) - Brief
overview of metabolomics versus transcriptomics and their added value ........................................ 17
Micro-Arrays ................................................................................................................................. 18
RNA-Seq ....................................................................................................................................... 18
DNA Sequencing ........................................................................................................................... 19
Metabolomics ................................................................................................................................ 20
Transcriptomics ............................................................................................................................. 20
Proteomics ..................................................................................................................................... 21
5) What is the advantage of transcriptomics over WES combined with Metabolomics? .............. 21
Case 2 – Identical or Not? ..................................................................................................................... 22
1) What is ALS? How does it come about? How heritable is ALS? Multifactorial stuff
(environmental effects): Short summary (mainly genetic basis) ....................................................... 22
ALS and Symptoms....................................................................................................................... 22
Causes............................................................................................................................................ 22
Pathophysiology ............................................................................................................................ 22
1
, 2) How can monozygosity or dizygosity of twins be determined? And how can genetically
identical twins have different phenotypes? (Partial heterochromia example in the case) ................. 23
Physical Features ........................................................................................................................... 23
Placenta-based examination .......................................................................................................... 23
DNA-Testing ................................................................................................................................. 23
Random Interesting Info ................................................................................................................ 24
Sectoral Heterochromia ................................................................................................................. 24
3) Which tests are online available for genetic testing? What technologies do the companies use
(especially the Twin-Testing Site and 23andMe)? What information do those techniques provide?
How much does it cost? → Direct consumer testing for health and/or “fun”.................................... 24
Types of DTC Genetic Test ........................................................................................................... 25
EasyDNA....................................................................................................................................... 25
23andMe ........................................................................................................................................ 25
Illumina Chips for Genotyping SNPs ............................................................................................ 26
4) Look into the lecture for cases/examples of WGS of monozygotic twins? What were these
research results? ................................................................................................................................ 26
Differences in Monozygotic Twins ............................................................................................... 26
5) Ethical and Legal aspects .......................................................................................................... 28
Ethical Considerations ................................................................................................................... 28
Legal Aspects ................................................................................................................................ 28
Workshop 2 – WES Analysis ................................................................................................................ 29
Journal Club 2 – Prime Editing ............................................................................................................. 30
Case 3 – Pick the Model that Suits You ................................................................................................ 32
1) What is Autosomal Recessive Cerebellar Ataxia (ARCA)?...................................................... 32
Cerebellar Ataxia ........................................................................................................................... 32
Ataxia and Possible Molecular Causes.......................................................................................... 32
2) Which in silico methods are available for predicting the genetical testing? a) What are their
advantages and disadvantages? ......................................................................................................... 34
Databases ....................................................................................................................................... 34
Software Programs and their Utility .............................................................................................. 34
Advantages .................................................................................................................................... 34
Disadvantages................................................................................................................................ 35
3) Which in vitro methods are available for genetic testing? a) What are their advantages and
disadvantages? b) What are their ethical concerns for such methods? ........................................... 35
Traditional in vitro cell cultures .................................................................................................... 35
3D Cultures and Organoids ........................................................................................................... 35
2
, 4) What are conventional animal models used within genetical research? (EUROPEAN
LEGISLATION: WAD?) a) What are their advantages and disadvantages? b) What are the ethical
concerns for each of them? ................................................................................................................ 36
3Rs ................................................................................................................................................. 36
Invertebrates ONLY DROSOPHILA HAS A GENOME-WIDE KNOCKDOWN
LIBRARY!!!! 36
Vertebrates..................................................................................................................................... 37
Pure and Applied Research ........................................................................................................... 38
Ethical Considerations ................................................................................................................... 38
Alternatives to Animal Testing ..................................................................................................... 39
(ARCA) Considerations ................................................................................................................ 39
5) Propose an experimental set-up for investigating the genetical findings within the case? In
Silico, in vitro, in vivo, what models can be specifically used in this case, and why or why not? ... 40
In Vitro cell tissue Notes ............................................................................................................... 40
Case 4 – ................................................................................................................................................. 41
1) What is known and what is not known about the human genome? ........................................... 41
Completeness of the Human Genome Sequence ........................................................................... 41
Protein Coding Genes (PCGs) ....................................................................................................... 41
Non-Coding DNA ......................................................................................................................... 42
2) What are the advantages and disadvantages of Forward Genetics approaches? Pick an Example
(JC Paper): What is Genetic Screening? ........................................................................................... 43
Forward Genetic Screening ........................................................................................................... 43
Reverse Genetic Screening ............................................................................................................ 45
Mapping Mutants .......................................................................................................................... 46
Journal Club Genetic Death-Screening ......................................................................................... 46
Laboratory Mouse Breeding .......................................................................................................... 46
2021 ............................................................................................................................................... 47
3) What does it look like in an animal model? .............................................................................. 47
4) Apply the knowledge to the case, focussing on nDNA (rather than mtDNA) .......................... 48
Presentation Notes and JC ..................................................................................................................... 50
Case 5 – ................................................................................................................................................. 52
1) What are proto-oncogenes and tumour suppressor genes (TSGs)? What are general treatments
for gain-of-function and loss-of-function treatments for cancers? .................................................... 52
General Treatments ....................................................................................................................... 53
2) What are current principles/techniques/drugs used to treat gain-of-function mutant proto-
oncogenes? ........................................................................................................................................ 53
Oncogene Addiction ...................................................................................................................... 53
3) Why can loss-of-function genes not be properly treated for cancer? ........................................ 55
3
, 4) What is synthetic lethality? And can it be abused for drug treatments for certain cancers? i.e.
BRCA ................................................................................................................................................ 57
Biological Importance ................................................................................................................... 57
5) How can you screen for synthetic lethality processes in cancer? And thereby find novel drug-
targets? Look for a BRCA paper ....................................................................................................... 58
Screening ....................................................................................................................................... 58
6) How can cancers become resilient against the PARP-inhibitor treatment in case of BRCA-
cancers? ............................................................................................................................................. 59
BRCA’s Achilles Heel .................................................................................................................. 60
PARP Inhibitor Principle............................................................................................................... 60
PARP-Inhibitor Resistance............................................................................................................ 60
Case 6 – ................................................................................................................................................. 61
1) What is the Krebs-Cycle and how is it involved within regulating epigenetic modifications? . 61
NADPH ......................................................................................................................................... 61
Cancer............................................................................................................................................ 61
Epigenetics .................................................................................................................................... 61
2) What are the pathophysiology and pathogenesis of Glioblastoma? .......................................... 63
Pathogenesis .................................................................................................................................. 63
3) What do IDH-1/2 normally do? And what goes wrong within neuroblastoma? ....................... 64
Isocitrate Dehydrogenase-1/2 (IDH1/2) ........................................................................................ 64
IDH1/2 in Cancer .......................................................................................................................... 66
4) What does MGMT normally do? And what goes wrong within neuroblastoma? ..................... 68
MGMT........................................................................................................................................... 68
Temozolomide ............................................................................................................................... 68
5) What is the current standard care for glioblastoma? Can IDH-1/2 and/or MGMT be used as
biomarkers for Glioblastomas? Are they viable therapeutic targets? ................................................ 69
Current Standards .......................................................................................................................... 69
Biomarkers .................................................................................................................................... 69
Therapeutic Targets ....................................................................................................................... 70
Journal Club 4 and Project stuff – Mutatome – ..................................................................................... 72
Case 7 – Cardiogenetics ........................................................................................................................ 74
1) What is Cardiac Disease and sudden cardiac death? ................................................................. 74
Epidemiology ................................................................................................................................ 74
Pathophysiology ............................................................................................................................ 74
2) What are the physiological differences between a normal and athlete’s heart? ........................ 75
Signs and Symptoms ..................................................................................................................... 75
Cause ............................................................................................................................................. 75
4
