Human Genetics Dr. Alter

1: Genome : numerical alteration of the complete genome or numerical alteration of a single chromosome

2: Chromosome : insertion, deletion, duplication, inversion, translocation

3: Gene : point mutation, mutations of several kilobases, mutations in regulatory sequences

- large scale vs. small scale - common vs. rare - pathogenic vs. non-pathogenic

abnormal number of chromosomes

e.g. Monosomy only one chromosome from a pair

• Triploidy three copies of every chromosome

• Tetraploidy four copies of every chromosome

• (Mosaicism two or more populations of cells with different genotypes)

• Trisomy three copies of (at least) one chromosome

Single nucleotide polymorphism

Polymorphism, greek: Polymorphismos, „diversity“ Polymorphisms are variations with a frequency > 1% in the population and are not expected to cause major phenotypic effects.

Copy number variation

… are structural variations (duplication or deletion).

… occur due to non-allelic homolgous recombination.

… are suprisingly frequent in human genome (~5%).

… are responsible for by far the greatest number of nucleotides that differ between two genomes.

… can cause diseases.

… play an important role in development of functionally new genes or extention of gene activity (i.e. olfactory receptorgenes).

= short tandem repeats

high genetic diversity (5 – 20 repeat units)

Trinucleotide Repeat Disorders (within a gene locus)

used for DNA profiling (cancer diagnostics, paternity testing, forensic identification)

• SNPs

• CNVs

• Microsatellites

• Failure in DNA repair

• Errors in recombination

• Errors in replication

SNPs

Causes of DNA damage

• External causes: [ionizing radiation, ultraviolet radiation, environmental chemicals] 

• Internal causes: [depurination, deamidation, reactive oxygen species (ROS), nonenzymatic methylation, normal DNA metabolism] 

- Failure in DNA repair

- Errors in recombination and replication

• Derived from a SNP 

• Exchange of the encoding amino acid leads to premature stop codon  

• Possible consequence truncated protein, protein degradation, NMD (nonsense mediated mRNA decay)

   

• exchange of an amino acid (missense-mutations)

• generation of a truncated protein (nonsense or frameshift mutation) or a different protein (splice-mutations)

• no or less protein is expressed (nonsense-, frameshift, splice-mutations, deletions, insertions, regulatory mutations)

• special-case: trinucleotide-disorders (e.g. Huntington disease)

• nature of the change

• allele frequency (MAF, minor allele frequency)

• functional studies

• pattern of inheritance

• the nature of the change • functional assays • penetrance and expressivity in dominant disorders • segregation within family • ethnic origin • other affected individuals

• silent mutation —> same amino acid (only in rare cases pathogenic) on wobble nucleotide

• missense mutation —> different amino acid (conservative or non conservative = same or different AA class, important if structure or function of protein is changed)

• nonsense mutation —> premature stop-codon (lead to truncated protein, protein degradation, NMD)

• readthrough mutation —> stop-codon is lost (extended Protein)

• Frameshift mutations —> Insertion or Deletion (usually leads to premature stop codon downstream and with that to degradation and NMD)

• no protein synthesis from such an allele

• Mutations in promoter e.g. TATA-Box —> affects expression level, timepoint, tissue

• Mutation in splice site —> splice donor, splice acceptor, branching site, splice enhancer, splice silencer —> leads to altered splicing efficiency, exon skipping, intron retention (not all pathogenic because alternative splicing is a natural variability

• Mutations in 5‘UTR —> e.g. 5‘Cap (stability of mRNA)

• Mutations in 3‘UTR —> e.g. polyadenylation site (stability and export of mRNA)

the same triplet is repeated far to often, there is a normal repeat number and the pathogenic one

e.g. Huntington disease (normal 9-35 vs. pathogenic 36-121)

in coding sequence > altered protein

in non-coding sequence > altered regulation

• Chromosomal diseases (diese Erkrankungen sind vorallem in der frühen Embryonalentwicklung wichtig und ist sehr häufig für spontan Abort (kommt in 50 % der Schwangerschaften vor)

• monogenetic diseases (kommen vorallem im Kindesalter auf) [20 / 1000]

• multifactorial diseases (kommen am häufigsten auf, da sie sich mit dem Alter entwickeln, Umwelteinflüsse sorgen für genetische Veränderungen und führen irgendwann zu Krankheiten —> Krebs) [646 / 1000}

• Epigenetic

• mitochondrial

• somatic [240 / 1000}

• autosomal recessive

• autosomal dominant

• X-chromosomal recessive/dominant

Further modes:

• mitochondrial • multifactorial • epigenetic

• autosomal recessive —> one affected allele is tolerated, affected individuals are homozygous or compound heterozygous (both genders, not in every generation)

  e.g. Cystic fibrosis (CF) (= Muskoviszidose) epithelial fluid transport is dysregulated because of disfunctional gene encoding for chloride channels

• autosomal dominant —> one affected allele is not tolerated, affected individuals are heterozygous (usually in every generation, usually affected individuals have also one affected parent, sporadic cases can be de novo)

  e.g. haploinsufficency (eine gute Kopie des Alleles ist nicht genug für gesunden Phenotyp… sehr selten), example Aniridia = absence of iris

  e.g. dominant negative effect —> Osteogenesis imperfecta = brittle bone disease (Glasknochen)

• A loss-of-function mutation is a genetic mutation that leads to a reduction or complete loss of the normal function of a gene -> no protein, non-functional protein, disrupted functional domain (wie bei Glasknochen hier wird die dicht gepackte, gleichmäßige Struktur durch die Mutation zerstört)

• Examples: Cystic fibrosis (CF), Hemophilia, Tay-Sachs disease 

Loss of function is very common in autosomal recessive inheritance

• constitutively active (missense-mutation)

• insert to regulatory signals (missense-mutation)

• expressed at the wrong time or tissue or amount (regulatory mutation)

Achondroplasia (= fehlende Knorpelbildung) (Peter Dinklage)

more common in autosomal dominant inheritance than in autosomal recessive inheritance

1. Library Preparation:

   • DNA or RNA is extracted from the sample of interest.

   • The DNA or RNA is fragmented into smaller pieces, and adapters are ligated to the ends of these fragments. These adapters contain sequences necessary for binding them to the surface of flow cell —> hybridisation.

2. Cluster Generation:

   • Fragments with adapters are amplified through polymerase chain reaction (PCR). —>

   • The amplified fragments are then attached to a solid surface, forming clusters of identical DNA fragments.

   • —> bridge building (millions of clusters)

   • —> washing away so only one direction strands are read (and in read 2 its the other strand e.g. first 3´-5´then 5´-3´)

3. Sequencing:

   • Various NGS platforms use different sequencing-by-synthesis methods.

   • Fluorescently labeled nucleotides are added one at a time, and as each nucleotide is incorporated into the growing DNA strand, a fluorescence signal is detected.

4. Data Analysis:

   • Raw data from the sequencing run is processed to generate sequence reads.

   • Bioinformatics tools are employed to align reads to a reference genome, identify variations, and interpret biological significance.

Advantages of Next Generation Sequencing (NGS):

1. High Throughput

2. Speed

3. Cost-Effective

4. Versatility

5. Sensitive Detection

Disadvantages of Next Generation Sequencing (NGS):

1. Read Length

2. Error Rates

3. Bioinformatics Challenges

4. Equipment Costs

5. Data Storage and Management