Lectures molecular basis of disease
College 0: Introduction molecular basis of diseases
In cystic fibrosis (CF) the cystic fibrosis transmembrane conductance regulator (CFTR), which is a Cl-
pump, is mutated. If CFTR does not work → water will not enter the cell → mucus stays thicker.
A sweat test is used to diagnose CF: there is an electrode with filter paper. If the filter paper absorbs
high amounts of chloride, there is a possible mutation in the CFTR.
Different mutations and their effects:
- Missense change: amino acid change due to a mutation in the RNA.
- Nonsense mutation: no amino acids are built in → the protein will end at the nonsense
mutation.
- Frameshift: the reading frame is shifted, and the shift is not in a multitude of 3 nucleotides.
- In-frameshift: the reading frame is shifted in a multitude of 3.
- Splicing defect: parts of the intron are included in the reading frame.
- Promotor defect: mutations in the promotor.
CTFR
The types of CTR defects are shown in the figure on the
right.
Treatment
Treatment options for CF-patients:
- Ataluren: build in any amino acid rather than
letting the protein stop.
- Corrector: make sure the proteins are folded correctly.
- Potentiator: makes the proteins more powerful → increased transport of Cl-.
- Stabilisator: prevents truncation of proteins.
- Orkambi: a synthetic drug consisting of 2 components:
o Ivacaftor: restores the chloride channel function.
o Lumacafor: facilitates CFTR folding.
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ID: Introduction
Variant: any deviation from the reference genome.
Polymorphism: ≤1 percent of the alleles in a population.
Mutation: <1 percent of the alleles in a population.
Pathogenic: disease-causing mutation or polymorphism.
CNV (copy number variant): deletion or duplication ≥1 kb.
The reference genome consists of six libraries, all consisting of one person. The reference genome is
the golden standard: DNA is compared to the reference genome.
Examples of genome browsers: UCSC and Ensembl.
Between two individuals 5982 million basepairs are the same and there will be a difference in about
18 million basepairs. Most difference occurs in the SNVs (single nucleotide variants).
Genome variations from small to big:
, 1. Single nucleotide variant (SNV) → basepair change.
2. Insertion or deletion (indel) → ≤10 bp.
3. Repeat expansion: 2 to >6000 basepairs. Repeats can form secondary RNA-structures (e.g.
loops), so they can expand or get smaller very easily.
4. Copy number variant (CNV): deletion or duplication >1000 bp.
5. Structural chromosomal abnormalities: translocations, inversions, etc.
6. Aneuploidy: duplication of one chromosome, e.g. Down syndrome.
The complete range of possibilities is not covered here, but purely based on technique: from next
generation sequencing (short range sequencing) to chromosomal microarrays. Genome sequencing
could be able to close this gap.
The dynamic genome: about five percent of the genome can differ between individuals.
Variant nomenclature
Main classes, groups and types of mutation and effects on protein product.
Class Group Type Effect on protein product
Substitution. Synonymous. Silent Same amino acid.
Non-synonymous. Missense Altered amino acid → may affect protein
function/stability.
Nonsense Stop codon → loss of function or
expression due to degradation of mRNA.
Splice site Aberrant splicing → exon skipping or
intron retention.
Promoter Altered gene expression.
Deletion. Multiple of 3 (codon). In-frame deletion of one or more amino
acid(s) → may affect protein function or
stability.
Not multiple of 3. Frameshift. Likely to result in premature termination
with loss of function or expression.
Large deletion. Partial gene May result in premature termination with
deletion. loss of function or expression.
Whole gene Loss of expression.
deletion.
Insertion. Multiple of 3 (codon). In-frame insertion of one or more amino
acid(s) → may affect protein function or
stability.
Not multiple of 3. Frameshift. Likely to result in premature termination
with loss of function or expression.
Large insertion. Partial gene May result in premature termination with
duplication. loss of function or expression.
Whole gene May have an effect due to increased gene
duplication. dosage.
Expansion of Dynamic Altered gene expression or altered
trinucleotide repeat. mutation. protein stability or function.
,Silent changes
Normal: ATG TAT TGG CGA ATG ACC.
Variant: ATG TAC TGG CGA ATG ACC.
TAT and TAC both code for tyr → c.6T>C; p.(=).
c stands for coding. p stands for protein: the protein does not change.
A silent change can cause a disease anyway, because the expression of the new protein can be
different.
Missense change
Normal: ATG TAT TGG CGA ATG ACC.
Variant: ATG CAT TGG CGA ATG ACC.
While TAT codes for tyr, CAT codes for his → c.4T>C; p.(tyr2His). The two stands for the fact that it is
the second amino acid.
Nonsense change
Normal: ATG TAT TGG CGA ATG ACC.
Variant: ATG TAT TGG TGA ATG ACC.
While CGA codes for arg, TGA codes for a stop-codon → c.10C.T; p.(Arg4*).
Frameshift change
Normal: ATG TAT TGG CGA ATG ACC
Variant: ATG TAT TGG CGA TGA CC
In this case, the deletion of an ATG codes for met, while TGA codes for a stop-codon →
c.12delA; p.(Met5fs*). Always consider the first duplication (first A in this case) to be deleted.
Insertion/duplication
Normal: ATG TAT TGG CGA ATG ACC.
Variant: ATG TAT TGG CGA AAT GAC.
May result in premature termination with loss of function or expression.
In this case, two times A turns into three times A: duplication of one A, or an insertion of one A?
ATG codes for met, while AAT codes for asn. Human genes have only one reading frame: if you lose
the reading frame, you will always encounter a stop-mutation within twenty amino acids. Write this
down using an * → c.12dupA; p.(Met5fs*).
In frame deletion
Normal: ATG TAT TGG CGA ATG ACC.
Variant: ATG TAT TGG ATG ACC.
Position 10 to 12 is deleted → c.10_12delCGA; p.(Arg4del).
Splice site changes
There are three exons (see figure on the right), with a
mutation for exon 2: CAG to CAA. The exon will be
skipped. Always start with the coding sequence →
c.33-1G>A; p.(?).
Basepair 33 is the first basepair in exon 2. If basepair 32 is
changed it moves to 33. Minus 1: on this position is a G
which is changed to an A.
Upon a promotor change, e.g. -100 (100 basepair before
the coding sequence starts).
When using exon sequencing, always take a bit of the
intron with it: canonical splice sites. These splice sites are vital.
Genome variation and disease
Rare alleles cause Mendelian disease → high effect on disease phenotype.
Common variants usually have a low effect on disease phenotype.
, Intellectual disability (ID)
Definition of ID
- Intelligence-quotient (IQ) < 70. 70 is 2 standard deviations below average.
- Limitations in adaptive functioning.
- Present before 18 years of age.
Classification of ID
- Profound: IQ <20 → institutionalised.
- Severe: IQ 20-34 → special education.
- Moderate: IQ 35-49 → special education.
- Mild: IQ 50-69 → normal education.
ID causes
Can be either multifactorial (culturofamilial) or monogenic (organic).
Mostly patients with ID look very normal. Most causes of ID are de novo mutations → 80% of cases
are de novo. Nowadays the IQ is more important than ever: society is very demanding. About 10% of
genes are involved in ID → high number of ID genes, so a high change of de novo mutations in these
genes.
ID patient groups
- Non-syndromic (nsID): only problems in IQ.
- Syndromic (sID): other problems apart from IQ, e.g. facial characteristics, microcephaly.
The genes causing nsID and sID are partially shared → genotype and phenotype overlap.
Identification of ID genes is important for diagnosis, counselling, therapy, and for science
(development and functioning of the brain). Therapy, however, is usually not a possibility after the
brain has alrady formed.
➔ Problem: genetically heterogeneous with around 2000 genes playing a role.
ID is a heterogeneous disorder.
- Allelic heterogeneity: same phenotype, same gene, different mutations.
- Locus heterogeneity: same phenotype, different genes. Locus means different position,
different position means different gene.
In the X-chromosome most ID genes are already found, while on the autosomes only half of the ID
genes are known.
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ID: CMA (chromosomal microarrays) ~ cytogenetic SNP array analysis
One of the main options for ID testing is microarrays.
Intellectual disability (IQ<70) occurs in 2% of the population. It is mostly diagnosed in children with
developmental backlog. When developmental milestones are not met, the child is sent to a
paediatrician. IQ testing is one of the first steps in diagnosis. Most children are not only suffering
from ID, but have more complex phenotype: e.g. facial dysmorphism, heart defect, etc.
Karyotyping
The first step in diagnosis before 2009 is karyotyping: looking at stained chromosomes through a
microscope. However, karyotyping takes a lot of time and training.
When a missing piece of chromosome can be spotted through the microscope: big ID problems.
Everything that can be spotted through the microscope are huge alterations.
