Table of Contents
Lecture – 1 – Organisation and Variation of the Human Genome .......................................................... 5
Case – 1 – Amazing Properties of the Human Genome ........................................................................ 11
1) What is the Human Genome Project? ....................................................................................... 11
2) What is the Human Karyotype and the chromosomes? Also focus on structural features ........ 11
Centromeres................................................................................................................................... 12
Replication Origins........................................................................................................................ 12
Telomeres ...................................................................................................................................... 12
General Genome ............................................................................................................................ 12
Karyotype ...................................................................................................................................... 12
3) Explain the basic structure of the human genome? - Protein Coding Genes - Non-Coding
RNAs - Mobile Elements (LINEs, SINEs, maybe triplet repeats) ................................................... 13
Protein-Coding Genes: Structures and Expression ........................................................................ 13
RNA Genes and Noncoding RNAs ............................................................................................... 14
Regulatory RNAs .......................................................................................................................... 15
Repetitive DNA in the Human Genome ........................................................................................ 16
Organisation of Gene Families ...................................................................................................... 16
Benefits of Gene Duplication ........................................................................................................ 17
Highly Repetitive Noncoding DNA in the Human Genome ......................................................... 17
4) What are all types of non-coding RNAs present within the genome? What are their functions
and their structures?........................................................................................................................... 18
Translation ..................................................................................................................................... 18
RNA-Splicing ................................................................................................................................ 19
DNA-Replication........................................................................................................................... 19
Gene-Regulation ............................................................................................................................ 19
Genome Defence ........................................................................................................................... 19
Chromosome Structure .................................................................................................................. 19
Bifunctional ncRNAs .................................................................................................................... 19
tRNA ............................................................................................................................................. 20
rRNA ............................................................................................................................................. 20
miRNA .......................................................................................................................................... 20
5) Comparative Genomics: What is the C-Value Paradox among different organisms and why can
this be considered a paradox; how could it possibly be explained? - Genome Size - Non-Coding
RNAs ................................................................................................................................................. 20
Identifying genes through Evolutionary Conservation .................................................................. 21
1
, The ENCODE Project ................................................................................................................... 21
Functional Significance of the Human Genome............................................................................ 21
6) Human nuclear DNA versus bacterial and mitochondrial DNA (is mtDNA different from
prokaryotic circular DNA) ................................................................................................................ 22
The Mitochondrial Genome: Economical Usage but Limited Autonomy..................................... 22
Gene Distribution in the Human Genome ..................................................................................... 23
Journal Club – 1 – The Origin of the Major Cystic Fibrosis Mutation ................................................. 24
Lecture – 2 – Gene Expression Regulation at Transcriptional and Translational Levels ..................... 27
Lecture – 3 – Nuclear Organisation of Gene Expression ...................................................................... 31
Case – 2 – The Origin and Fate of Genetic Mutations .......................................................................... 35
1) What are the type of Mutation that may occur how detrimental can their effects be? How to
these mutations arise at a molecular Level? ...................................................................................... 35
Causes of Mutations ...................................................................................................................... 35
Mutation Type Classification ........................................................................................................ 37
2) What are the differences between Single Occurrence and Recurrent Mutations? How can these
be diagnosed; how do we know it was a single occurrence or recurrent? ......................................... 39
Linkage Disequilibrium ................................................................................................................. 40
Incomplete Sweep ......................................................................................................................... 40
Microsatellite and Haplogroup Determination .............................................................................. 41
3) What is the Hardy-Weinberg Equation and does the Equilibrium indicate? In which cases may
HWE be used, and in which cases may it not be applied? (What are the conditions that are required
to apply this equilibrium?) Apply it to real cases .............................................................................. 41
Bi-Allelic ....................................................................................................................................... 41
Poly-Allelic ................................................................................................................................... 42
Balancing Selection ....................................................................................................................... 42
4) What do the mutations do? How does it cause this disease specifically? What is their
frequency? And how can it have reached this frequency? - Factor V Leiden: R506Q - -13910C>T;
Selective Sweep - ΔF508; Balancing Selection ............................................................................... 43
Venous Thrombosis – Factor V Leiden......................................................................................... 43
Lactose Intolerance – -13,910C>T ................................................................................................ 44
Cystic Fibrosis – ΔF508 ................................................................................................................ 45
5) Answer the questions specifically in each A), B) and C) .......................................................... 46
6) What mechanisms underlie the cause of spreading of such diseases?....................................... 46
Lecture – 4 – Organisation and Variation of the Human Genome ........................................................ 47
Lecture – 5 – Evolution and Population Genetics of Variation ............................................................. 49
Case – 3 – F5 Mutational Spectrum ...................................................................................................... 53
1) Mutations present in the CDS a) Does the mutation change the polypeptide sequence;
Synonymous, Non-Synonymous, Nonsense b) Frameshift Mutations c) Splicing Mutations ......... 53
2
, Small-Scale Mutations .................................................................................................................. 53
Impact on the Protein Sequence .................................................................................................... 53
Genesis and Frequency of Pathogenic Point Mutations ................................................................ 56
Surveying and Curating Point Mutations that cause Diseases....................................................... 56
2) Mutations present in the outside of the CDS a) Introns b) Cis-Regulatory Sequences:
Promotor, Enhancer, Silencer c) 5’-UTR and 3’-UTRs ................................................................... 58
Introns............................................................................................................................................ 58
Cis-Regulatory Sequences ............................................................................................................. 58
5’- and 3’-Untranslated Region (UTR) ......................................................................................... 59
3) Little Hardy Weinberg Equation Test ....................................................................................... 61
4) Answer Peter’s Questions.......................................................................................................... 61
What Missense Mutations are deleterious? ................................................................................... 61
Are all splicing mutations detrimental? Can Intronic Mutations cause a Phenotype? Can
Synonymous Mutations cause a phenotype ................................................................................... 62
What are the predicted Consequences of the Large Deletion in Patient #8? ................................. 62
What are the predicted Consequences of the Promotor Mutation Patient #9? Is this single
mutation enough to reduce the FV levels below 5%? ................................................................... 62
How is it possible that only one mutation was encountered in patient #3? ................................... 62
What about Patient #10?................................................................................................................ 63
Journal Club – 2 – ................................................................................................................................. 64
Case – 4 – Validation of variants of unknown significance .................................................................. 66
1) Repeat PCR and Molecular Cloning Principles ........................................................................ 66
PCR ............................................................................................................................................... 66
Cloning Principles ......................................................................................................................... 67
2) How can a specific mutation be induced within a DNA molecule? (Site-Directed Mutagenesis
(SDM)) - (Two Approaches: CRISPR-Cas vs External Mutation) ................................................... 68
Site-Directed Mutagenesis............................................................................................................. 68
3) Explain and Describe the methods mentioned in the Brain Storm (possibly look for other ones
as well): - Luciferase Reporter Assay, Databases/Computational Modelling (Homology Modelling
and De Novo), RT-PCR, NMR, XRC, Recombinant Expression, Antibody and Expression
Characterisation, Mini-Gene Systems, etc. ....................................................................................... 71
Luciferase Reporter Assay or Fluorescent Protein ........................................................................ 71
Computational Modelling (Or just prediction with PolyPhen or SIFT) .................................... 71
Protein Structure Determination .................................................................................................... 74
Protein Functionality Determination ............................................................................................. 76
Mini-Gene Systems ....................................................................................................................... 79
4) Which methods/approaches would be most applicable to every type of mutation? Make a list to
address all mutations with their respective method. - Promotor Variants (Luciferase Report, and?)
3
, -Missense/Nonsense (WT, Mutagenesis (SDM), recombinant, anti-body and expression
characterisation) (((- Missense/Nonsense Less Important analysis with NMR, XRC))) - Alternative
Splicing: mRNA Reverse-Transcription PCR (purify mRNA from patients; Build a Model with
Mini-Gene Systems; check reference list) ......................................................................................... 81
Case – 5 – .............................................................................................................................................. 82
1) What are multifactorial diseases and how can you recognise them? - Name some examples -
Primarily Genetic Architecture.......................................................................................................... 82
2) What is Heritability? And what is missing Heritability? ........................................................... 84
3) What techniques are there to diagnose Multifactorial Diseases? How can GWAS studies be
used in the diagnosis? How does GWAS work? What are the issues? What type of results does it
provide? How are the results interpreted? ......................................................................................... 86
4) What is a Linkage Study? How is a Linkage Analysis performed? What results are gathered?
What are the problems? Etc............................................................................................................... 88
5) What is meant with Targeted Resequencing Projects? And what would be worthwhile
investigated? ...................................................................................................................................... 90
6) How can Non-Coding Regions of the genome contribute to Multifactorial Diseases? ............. 91
7) What other factors play a role in Multifactorial Diseases, besides genetics? - Epigenetics ..... 92
8) XXX .............................................................................................. Error! Bookmark not defined.
Lecture – 6 – Heritability and Multifactorial Treats.............................................................................. 93
Lecture – 7 – ........................................................................................................................................ 100
Case – 5 – ............................................................................................................................................ 100
Career Lecture – 1 – ............................................................................................................................ 117
Career Lecture – 2 – ............................................................................................................................ 118
4
Lecture – 1 – Organisation and Variation of the Human Genome .......................................................... 5
Case – 1 – Amazing Properties of the Human Genome ........................................................................ 11
1) What is the Human Genome Project? ....................................................................................... 11
2) What is the Human Karyotype and the chromosomes? Also focus on structural features ........ 11
Centromeres................................................................................................................................... 12
Replication Origins........................................................................................................................ 12
Telomeres ...................................................................................................................................... 12
General Genome ............................................................................................................................ 12
Karyotype ...................................................................................................................................... 12
3) Explain the basic structure of the human genome? - Protein Coding Genes - Non-Coding
RNAs - Mobile Elements (LINEs, SINEs, maybe triplet repeats) ................................................... 13
Protein-Coding Genes: Structures and Expression ........................................................................ 13
RNA Genes and Noncoding RNAs ............................................................................................... 14
Regulatory RNAs .......................................................................................................................... 15
Repetitive DNA in the Human Genome ........................................................................................ 16
Organisation of Gene Families ...................................................................................................... 16
Benefits of Gene Duplication ........................................................................................................ 17
Highly Repetitive Noncoding DNA in the Human Genome ......................................................... 17
4) What are all types of non-coding RNAs present within the genome? What are their functions
and their structures?........................................................................................................................... 18
Translation ..................................................................................................................................... 18
RNA-Splicing ................................................................................................................................ 19
DNA-Replication........................................................................................................................... 19
Gene-Regulation ............................................................................................................................ 19
Genome Defence ........................................................................................................................... 19
Chromosome Structure .................................................................................................................. 19
Bifunctional ncRNAs .................................................................................................................... 19
tRNA ............................................................................................................................................. 20
rRNA ............................................................................................................................................. 20
miRNA .......................................................................................................................................... 20
5) Comparative Genomics: What is the C-Value Paradox among different organisms and why can
this be considered a paradox; how could it possibly be explained? - Genome Size - Non-Coding
RNAs ................................................................................................................................................. 20
Identifying genes through Evolutionary Conservation .................................................................. 21
1
, The ENCODE Project ................................................................................................................... 21
Functional Significance of the Human Genome............................................................................ 21
6) Human nuclear DNA versus bacterial and mitochondrial DNA (is mtDNA different from
prokaryotic circular DNA) ................................................................................................................ 22
The Mitochondrial Genome: Economical Usage but Limited Autonomy..................................... 22
Gene Distribution in the Human Genome ..................................................................................... 23
Journal Club – 1 – The Origin of the Major Cystic Fibrosis Mutation ................................................. 24
Lecture – 2 – Gene Expression Regulation at Transcriptional and Translational Levels ..................... 27
Lecture – 3 – Nuclear Organisation of Gene Expression ...................................................................... 31
Case – 2 – The Origin and Fate of Genetic Mutations .......................................................................... 35
1) What are the type of Mutation that may occur how detrimental can their effects be? How to
these mutations arise at a molecular Level? ...................................................................................... 35
Causes of Mutations ...................................................................................................................... 35
Mutation Type Classification ........................................................................................................ 37
2) What are the differences between Single Occurrence and Recurrent Mutations? How can these
be diagnosed; how do we know it was a single occurrence or recurrent? ......................................... 39
Linkage Disequilibrium ................................................................................................................. 40
Incomplete Sweep ......................................................................................................................... 40
Microsatellite and Haplogroup Determination .............................................................................. 41
3) What is the Hardy-Weinberg Equation and does the Equilibrium indicate? In which cases may
HWE be used, and in which cases may it not be applied? (What are the conditions that are required
to apply this equilibrium?) Apply it to real cases .............................................................................. 41
Bi-Allelic ....................................................................................................................................... 41
Poly-Allelic ................................................................................................................................... 42
Balancing Selection ....................................................................................................................... 42
4) What do the mutations do? How does it cause this disease specifically? What is their
frequency? And how can it have reached this frequency? - Factor V Leiden: R506Q - -13910C>T;
Selective Sweep - ΔF508; Balancing Selection ............................................................................... 43
Venous Thrombosis – Factor V Leiden......................................................................................... 43
Lactose Intolerance – -13,910C>T ................................................................................................ 44
Cystic Fibrosis – ΔF508 ................................................................................................................ 45
5) Answer the questions specifically in each A), B) and C) .......................................................... 46
6) What mechanisms underlie the cause of spreading of such diseases?....................................... 46
Lecture – 4 – Organisation and Variation of the Human Genome ........................................................ 47
Lecture – 5 – Evolution and Population Genetics of Variation ............................................................. 49
Case – 3 – F5 Mutational Spectrum ...................................................................................................... 53
1) Mutations present in the CDS a) Does the mutation change the polypeptide sequence;
Synonymous, Non-Synonymous, Nonsense b) Frameshift Mutations c) Splicing Mutations ......... 53
2
, Small-Scale Mutations .................................................................................................................. 53
Impact on the Protein Sequence .................................................................................................... 53
Genesis and Frequency of Pathogenic Point Mutations ................................................................ 56
Surveying and Curating Point Mutations that cause Diseases....................................................... 56
2) Mutations present in the outside of the CDS a) Introns b) Cis-Regulatory Sequences:
Promotor, Enhancer, Silencer c) 5’-UTR and 3’-UTRs ................................................................... 58
Introns............................................................................................................................................ 58
Cis-Regulatory Sequences ............................................................................................................. 58
5’- and 3’-Untranslated Region (UTR) ......................................................................................... 59
3) Little Hardy Weinberg Equation Test ....................................................................................... 61
4) Answer Peter’s Questions.......................................................................................................... 61
What Missense Mutations are deleterious? ................................................................................... 61
Are all splicing mutations detrimental? Can Intronic Mutations cause a Phenotype? Can
Synonymous Mutations cause a phenotype ................................................................................... 62
What are the predicted Consequences of the Large Deletion in Patient #8? ................................. 62
What are the predicted Consequences of the Promotor Mutation Patient #9? Is this single
mutation enough to reduce the FV levels below 5%? ................................................................... 62
How is it possible that only one mutation was encountered in patient #3? ................................... 62
What about Patient #10?................................................................................................................ 63
Journal Club – 2 – ................................................................................................................................. 64
Case – 4 – Validation of variants of unknown significance .................................................................. 66
1) Repeat PCR and Molecular Cloning Principles ........................................................................ 66
PCR ............................................................................................................................................... 66
Cloning Principles ......................................................................................................................... 67
2) How can a specific mutation be induced within a DNA molecule? (Site-Directed Mutagenesis
(SDM)) - (Two Approaches: CRISPR-Cas vs External Mutation) ................................................... 68
Site-Directed Mutagenesis............................................................................................................. 68
3) Explain and Describe the methods mentioned in the Brain Storm (possibly look for other ones
as well): - Luciferase Reporter Assay, Databases/Computational Modelling (Homology Modelling
and De Novo), RT-PCR, NMR, XRC, Recombinant Expression, Antibody and Expression
Characterisation, Mini-Gene Systems, etc. ....................................................................................... 71
Luciferase Reporter Assay or Fluorescent Protein ........................................................................ 71
Computational Modelling (Or just prediction with PolyPhen or SIFT) .................................... 71
Protein Structure Determination .................................................................................................... 74
Protein Functionality Determination ............................................................................................. 76
Mini-Gene Systems ....................................................................................................................... 79
4) Which methods/approaches would be most applicable to every type of mutation? Make a list to
address all mutations with their respective method. - Promotor Variants (Luciferase Report, and?)
3
, -Missense/Nonsense (WT, Mutagenesis (SDM), recombinant, anti-body and expression
characterisation) (((- Missense/Nonsense Less Important analysis with NMR, XRC))) - Alternative
Splicing: mRNA Reverse-Transcription PCR (purify mRNA from patients; Build a Model with
Mini-Gene Systems; check reference list) ......................................................................................... 81
Case – 5 – .............................................................................................................................................. 82
1) What are multifactorial diseases and how can you recognise them? - Name some examples -
Primarily Genetic Architecture.......................................................................................................... 82
2) What is Heritability? And what is missing Heritability? ........................................................... 84
3) What techniques are there to diagnose Multifactorial Diseases? How can GWAS studies be
used in the diagnosis? How does GWAS work? What are the issues? What type of results does it
provide? How are the results interpreted? ......................................................................................... 86
4) What is a Linkage Study? How is a Linkage Analysis performed? What results are gathered?
What are the problems? Etc............................................................................................................... 88
5) What is meant with Targeted Resequencing Projects? And what would be worthwhile
investigated? ...................................................................................................................................... 90
6) How can Non-Coding Regions of the genome contribute to Multifactorial Diseases? ............. 91
7) What other factors play a role in Multifactorial Diseases, besides genetics? - Epigenetics ..... 92
8) XXX .............................................................................................. Error! Bookmark not defined.
Lecture – 6 – Heritability and Multifactorial Treats.............................................................................. 93
Lecture – 7 – ........................................................................................................................................ 100
Case – 5 – ............................................................................................................................................ 100
Career Lecture – 1 – ............................................................................................................................ 117
Career Lecture – 2 – ............................................................................................................................ 118
4
