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Molecular basis of diseases lecture notes
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Lecture notes of 33 pages for the course NWI-MOL055 at RU
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Molecular Basis of DiseasesMonday, 31st January
Introduction to Human Molecular Genetics
In patients with cystic fibrosis, the CFTR (cystic fibrosis transmembrane conductance
regulator) transports the Cl out of the cell. It also pulls sodium outside of the cell NaCl is
formed water is attracted fluid mucus. If this does not work then mucus gets thick and
you get CF.
Diagnostics: higher concentration of Cl in the sweat of the patient. (opposite of the lungs)
The main difference in Cl transport between the lungs and the sweat gland is that in one it is
secreted while in the other one it is absorbed.
Missense and nonsense mutations (in ORF), frameshift and deletion and splice sites (not
promotor sequences or deep intronic variants) are analyzed by exome sequencing.
Classification of CTRF mutations:
- No protein at all (stop mutation p.Gly542)
- No traffic (protein stuck in Golgi)
- No function (reaches the membrane but doesn’t get through)
- Less function (stop mutation, wrongly folded channel)
- Less protein
- Less stable
Treatment: Orkambi Ivacaftor restores Cl channel function and Lumacaftor facilitates
CFTR folding
Cl is also transported by SLC26A9! (solute carrier family) It makes sure that Ivacaftor doesn’t
work and influences the medication.
For a CFTR nonsense variant the best treatment is Ataluren.
Genomic Variation
Variation: any deviation from the reference genome
Polymorphism: variation > or = 1% of the alleles in a population (so we need 50 persons to
determine that)
Mutation: variation <1% of the alleles in a population
Pathogenic: disease-casing mutation or polymorphism
CNV (copy number variation): deletion or duplication > or = 1 kb
- Single nucleotide variant (SNV) base pair change (exome sequencing)
- Insertion or deletion < or = 10 bp (exome sequencing)
- Repeat expansion 2 to >6000 base pairs
, - Copy number variant (CNV), deletion or duplication
- Structural chromosomal abnormalities, translocations, inversions…
- Aneuploidy, one chromosome extra
Class Group Type Effect on protein product
Substitution Synonymous Silent Same amino acid
Non-synonymous Missense Altered amino acid
Nonsense (may affect protein
Splice site function or stability)
Promoter Stop codon (loss
of function)
Aberrant splicing-
exon skipping or intron
retention
Altered gene
expression
Deletion Multiple of 3 In-frame deletion
(codon) of one or more amino
Not multiple of 3 Frameshift acids
Large deletion Partial gene Likely to result in
deletion premature termination
Whole gene with loss of function or
deletion expression
May result in
premature termination
with loss of function or
expression
Loss of expression
Insertion Multiple of 3 In-frame insertion
Not multiple of 3 Frameshift of one or more amino
Large insertion Partial gene acids
Expansion of duplication Likely to result in
trinucleotide repeat Whole gene premature termination
duplication with loss of function or
Dynamic mutation expression
May result in
premature termination
with loss of function or
expression
May have an
effect due to increased
gene dosage
Altered gene
expression or altered
protein stability or
function
Silent changes: protein stays the same e.g., c.6T>C; p.(=)
Missense change: incorporation of wrong amino acid e.g., c.4T>C; p.(Tyr2His)
,Nonsense change: appearance of stop codon e.g., c.10C>T; p.(Arg4*)
Frame shift deletion: it is always believed to be the first one in case of two same amino acids
in a row e.g., c.12delA; p.(Arg4fs*)
Duplication: e.g., c.12dupA; p.(Met5fs*)
In frame deletion/insertion: c.10_12delCGA; p.(Arg4del)
Splice site changes: e.g., c.33-1G>Al p.(?)
Promotor sequence: c.1-333C>T
- P:protein
- G:genomic
- R:RNA
- C: coding
Intellectual disabilities: CMA/NGS
Intellectual disabilities (ID):
- It affects 2% of the population
- Often due to chromosomal abnormalities
- intelligence quotient <70, limitations in adaptive functioning, present <18 years old
- 4 classes: profound(<20), severe(20-34), moderate(35-49), mild(50-69)
- Inheritance can differ: autosomal dominant (de novo mutation), autosomal
recessive, X-linked
- Largely heterogeneous disease
CNVs are detected by karyotyping and chromosomal microarray
SNVs, Indels and CNVs are detected by exome sequencing and genome sequencing
Karyotyping: genome wide testing BUT: you only see abnormalities when they involve
millions of nucleotides. It is time consuming, difficult to automate, interpretation is
subjective (used for big aberrations)
Some patients that have clinical features that are very specific so they lead you to analysis of
a specific region. For example, people with down syndrome so only analyze chromosome 21.
However, some syndromes are more rare.
FISH (fluorescence in situ hybridization): more accurate method especially for small
aberrations.
Chromosomal microarrays
(CMA)
A glass slide with a large quantity of (FISH)
probes
DNA is isolated from the patient’s blood
sample. The DNA is labeled with a green
, fluorescence dye. Reference DNA is labeled with a red fluorescence dye. These are
hybridized to a FISH probe overnight. The red and green signals are analyzed.
This method is now used instead of karyotyping and FISH.
Example of SNV: moderate ID, hypertelorism (abnormal distance between two bodily parts),
ASD (heart defect), syndactyly, cutis marmorata (skin disorder with pinkish/blueish marble
appearance) deletion in chromosome 7. Could be de novo, that’s why parental DNA must
be checked. Every patient-parent trio is checked for Mendelian inheritance consistency.
Next Generation Sequencing (NGS)
also called massive parallel sequencing
Exome sequencing (for identifying new disease genes, for diagnosing patients and
for de novo mutations)
We are genetically 99.5% the same! But our DNA has 6.000.000.000 bases so a lot of
variations can happen.
Exome sequencing: reading of all coding regions of the genome. (~50 mil base pairs). You
take the DNA of the individual and you fish out the coding regions using enrichment
methods.
Advantage: cheaper and faster (~2 days)
- First up the DNA is fragmented into small pieces and the important ones are fished
out. The fragments are aligned using a reference genome.
- The second step is finding the differences (variant calling) comparing to the
reference genome.
- Finally with variant annotation information is collected about the variants and
interpretations about where the variant is relevant to a disease or not. If the
interpretation matches the phenotype, then we have diagnosis.
Coverage: the number of times a single position on the DNA has been interrogated by a
sequence reads.
Why do we need it?
- There are two alleles that we sequence at random, we need to be sure that we see
each allele at least once. (at least 20 reads)
- We want to be able to distinguish variants from sequencing errors.
- Some regions don’t enrich very well, if we sequence more, we will have a higher
chance of sequencing these regions as well.
Exome sequencing for disease gene identification:
Prioritization of variants causing genetic disease: private, non-synonymous variants (~50-
150). They appear on the patient but not on everyone else.
Example: Sensenbrenner syndrome
- Autosomal recessive disorder
- Ectodermal and skeletal abnormalities
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