Summary of all colleges of molecular basis of diseases with images and extra information. Extensive text and easy to read and understand. It may look like the summary does not have many pages, this is because the font size is reduced and small images have been added.
Human molecular genetics
In the last few years, the influence of genetics on modern health care has increased tremendously. Genetics has become essential for diagnosis,
prognosis, treatment and prediction of human disease.
Cystic fibrosis
Cystic fibrosis is a progressive, genetic disease that causes persistent lung infections and limits the
ability to breath overtime. In people with CF mutations in the cystic fibrosis transmembrane
conductance regulator gene causes the CFTR protein to become dysfunctional.
In normal lung there is an a airway wall and thin layer with mucus patients with CF have a thick
mucus making it difficult to breath and predisposing them to infections. In CF multiple organs are
infected like sinuses, lung, skin (sweat glands), pancreas, intestines and reproductive organs.
Immunoreactive trypsinogen is a blood test that measures the level of
immunoreactive trypsinogen, this is produced in the pancreas and
transported to the intestine where it is activated to form the enzyme
trypsin. In CF, thick mucus can obstruct pancreatic ducts and prevent
trypsinogen from reaching the intestine. Blood IRT levels are elevated
in people with CF but positive test results must be followed by sweat
chloride testing.
Sweat chloride testing involves measuring the chloride level in a
sweat sample. The sweat of a person with CF may be up to five times
saltier than normal. In a normal sweat gland the chloride
concentration is the same as the serum and thus isotonic.
CFTR channels release chloride out of cells and into serum because we need enough salt in blood thus the cell becomes hypotonic.
In a CF sweat gland the serum begins isotonic but the CFTR is abnormal and there is less chloride released so it is slightly hypotonic to isotonic.
If the CFTR does not work, chloride concentration inside the cell is higher. This causes increased water retention in cells, corresponding
dehydration of the extracellular space and an associated cascade of effects on various parts of the body like a thicker mucous membrane.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein and chloride channel in
vertebrates that is encoded by the CFTR gene. The gene is found on chromosome 7, on the long arm at
position q31.2 and has about 27 exons.
One of the most common variant is
p.Phe508del. In DNA diagnostics they check
30 of the 2000 most common variants.
Wildtype CFTR: normal transport of
chloride to the outside of the cell.
Stop mutation: There is no CFTR protein made.
Deletion 508F: protein gets stuck when it is made in the ER.
Mutation: proteins do enter transmembrane but do not transport.
Missense: can result in less function, less protein and less stable.
Most diseases are
caused by the classes I and II. Mutation type premature termination codon (PTC) means
there is a nonsense mutation that leads to a stop *.
Ataluren makes ribosomes less sensitive to premature stop codons by promoting
insertion of certain near-cognate tRNA at the site of nonsense codons. A corrector
pushes the effect of the channel protein to transport chloride better.
When a mutation causes loss of chloride conduction ivacaftor restores chloride
channel function. Ivacaftor is primarily used in the G551D mutation.
Orkambi combines ivacaftor with lumacaftor and is used to treat people with CF who
have two copies of the F508del mutation (facilitates CFTR folding and acts as a
chaperone).
FFV is a test that lest you breath out as much as you can, this test shows that
ivacaftor will work better with the TC variant.
SLC26A9 is an epithelial chloride channel that is expressed in several organs affected
in patients with cystic fibrosis. It is a potential alternative target to circumvent the
basic ion transport defect caused by deficient CFTR-mediated chloride transport in
CF.
When CFTR does not work SLC26A9 makes sure chloride is still excreted out of the cell. A modifier gene alters the expression of a human gene.
Genomic variation We all have 2 alleles, if you test 50 persons you have 100 alleles. The
Variation: any deviation from the reference genome. carrier frequency can be different from the allele frequency. The carrier
Polymorphism: variation in >1% of the alleles in population frequency is the proportion of individuals in a population who have a
single copy of a specific recessive genetic variant. Allele frequency is the
Mutation: variation in <1% of the alleles in population
relative frequency of an allele at a particular locus in a population,
Pathogenic: disease-causing mutation or polymorphism expressed as a fraction or percentage. It is the fraction of all
CNV: deletion or duplication >1kb chromosomes in the population that carry that allele.
,Missense: Change of a single base pair causes the
substitution of a different amino acid in the resulting
protein. May have no effect or it may render the protein
nonfunctional.
Nonsense: substitution of a single base pair that leads to
the appearance of a stop codon. Results in production of a
shortened, nonfunctional protein.
Frameshift: involves an insertion or deletion of nucleotide
in which the number of deleted base pairs is not divisible by
three. The entire DNA sequence following will be read
incorrectly.
In frame indel: an insertion or deletion in which the number
of base pairs is divisible by three.
Splicing: results in loss of entire exons
Promotor changes: change in expression.
The reference genome consists of only 6 persons that are sequenced. This is the golden
standard, 70% of this is only 1 person. The reference genome contains 3 billion base pairs,
these are stored in genome browsers like UCSC.
With sequencing you can easily find SNVs and indel variation. Repeat expansion often consist
of G and C, these are not separated easily because they are connected by three H-bonds
versus 2 bonds for A-T. You can find CNV with sequencing depending on how many reads you
have. Structural chromosomal abnormalities is when chromosomes are exchanged at the
break point. Aneuploidy means having a chromosome extra.
Within introns, a donor site (5’ end of the intron), a branch site and an acceptor site (3’ end of the intron) are
required for splicing. The splice donor site includes an almost invariant sequence GU at the 5’ end of the intron,
within a larger, less highly conserved region. The splice acceptor site at the 3’ end of the intron terminates the
intron with an almost invariant AG sequence.
Intellectual disability
Intellectual disability (ID) is a generalized neurodevelopmental disorder characterized by significantly
impaired intellectual and adaptive functioning. It is defined by an IQ <70. In addition to deficits in two or
more adaptive behaviors that affect every day and general living.
2.1% of people have a mild case of ID. 0.4% have a profound, severe or moderate case of ID. The cause of ID
is often unclear. With inheritance pattern it is seen that the disease can be recessive (both of parent one effected allele), de novo or it can lay
on the X chromosome. The use of genome wide detection of variation can find out which gene is responsible like karyotyping and chromosomal
microarray to find CNVs and exome and genome sequencing to find SNVs, indels and CNVs.
Intellectual disability is a heterogenous condition, this is one where the same disease or condition can be caused, or contributed to by varying
different genes or alleles. This can be broken down into allelic heterogeneity, when different variants at a single gene locus cause the same or
similar phenotypic expressions of a disease or condition. And locus heterogeneity, when variants at different gene loci cause the same or similar
phenotypic expressions of a disease or condition. In ID about
1000 ID genes are identified, some 1000 more to be expected. In The original idea was that you have one gene that is responsible for one phenotype
(CFTR). This only happens for few diseases and even then the phenotype is not the same.
ID there is a comorbidity of other defects, the same gene that In ID is even more difficult, you have thousands genes that cause ID and multiple
causes ID can also cause autism spectrum disorder. phenotypes. The genes do not work on their own but have a gene network.
Lumpers and splitter are opposing factions in any discipline that has to place individual examples into rigorously defined categories. A lumper is
an induvial who takes a gestalt view of a definition, all intellectual disabilities are the same. A splitter is one who takes precise definitions and
creates new categories to classify the diseases.
Brain
The first step in brain development is proliferation, if there is a problem there you expect people
have less brain cells. Then comes differentiation, normal cells are formed into neuroblast. Then
migration of where the cell is supposed to be. Functional incorporation means that the cells are at
the right place and then you have synapse formation, post and pre-synapse sends signals to the
brain.
At synapses there is neurotransmission. A neuron consist of a number of spines, in a patient this is
longer because they don’t encounter connection. In normal situation you have an action
potential and presynaptic processes (these say to the neurotransmitters that they are going to
be released). The neurotransmitters are used by detectors like sodium and calcium channels in
the postsynaptic density. After this you get cytoskeleton changes and signaling molecules.
Signaling molecules tell what is going to happen and tell to transcribe a gene into a protein
because that is needed in postsynaptic density.
Neurons communicate with each other via electrical events called action potentials and
chemical neurotransmitters. At the junction between two neurons (synaps), an action potential
causes neuron A to release a chemical neurotransmitter.
, In the synapses there are multiple pathways involved in ID. In purple are the
genes that are mutated and involved in ID. PSD proteins is the target of
neurotransmitters, this is where the signal comes into the cell. These proteins
transfer the signal into Rho GTPases, these give signal to actin polymerization
(assembly of endocytic vesicles). Proteolysis is the breakdown of proteins into
smaller polypeptides or amino acids, this happens in certain aspects in this
pathway. The Ras-MAPK signaling pathway is involved in epigenetic regulation,
they go into the nucleus and regulate transcription.
FMR1 causes fragile x-syndrome and is heavily studied.
Epigenetic regulation
Epigenetic regulation constrain expression by adapting regions of the genome to
maintain either gene silencing or gene activity. This is achieved through direct
chemical modification of the DNA region itself and by modification of proteins that are closely associated with the locus.
Epigenetic modifications are histone modification, RNA interference (miRNA that binds to mRNA, the
RNA is degraded or it inhibits translation) and DNA methylation (adding a methyl group to C).
There are 55 ID genes that are epigenetic regulators that are involved in writing, erasing and reading.
There are quite some genes, like EHMT1 that is a histone modification.
Mutations in EHMT1 cause kleefstra syndrome. Core phenotypes are DD/ID,
childhood hypotonia, facial characteristics. Various features are
micro/brachycephaly, obesity, heart defects, renal defects, seizure, behavioral
problems, autism and heario/vision loss.
A nucleosome is the basic structure unit of DNA
packaging in eukaryotes. The structure of a
nucleosome consist of DNA wound around eight
histone proteins and resembles thread wrapped around a spool. The four histones have
different protein chains, especially on N-terminus. The lysine can be modified, like adding
acetyl or methyl group. A serine can be phosphorylated. Thus a number of this amino acid
residues can be modified. EHMT1 methylates lysine 9. EHMT: histone 3 lysine 9 mono/di-
methylation. By methylation of lysine 9, the DNA gets more tightly wound around the histone
and thereby represses transcription.
75% that look like kleefstra syndrome you don’t find mutation in EHMT1 but other mutations.
By literature search you find out all these genes are involved in
the chromatic changing complex. Repressive mark negative regulates
EHMT1 usually downregulate the expression. transcription. It turns genes off.
NR1I3 is a constitutive androstane receptor and a key regulator of drug metabolism in liver (but also expression in brain). It dimerizes with
retinoic X receptor. To the hormone responsive element dimerize two protein and bind to HRE, then there is activating signal cointegrator-2
complex that bind to this complex. It increases gene expression.
MML3 is tri-methylation H3K4 (activation signal). It is in ASCOM complex. It is involved in NR-mediated
transactivation. MLL3 binds to ASCOM and you get a higher gene expression.
MML4 binds in the same way as MML3. This is a homozygous changes. You see that parents that are blood
related often have this mutation. MLL4 is a substitute of MML3.
SMARCB1 is Swi/Snf ATP-ase chromatin remodeling complex and is also involved in ASC complex.
Mutant flies An intellectual disabled fly will keep jumping on a female fly even after she
Are EHMT1 and MBD5 is ASCOM complex? Strategy: modeling ID in the fly pushed him away already.A functional interaction is one in which two proteins
are involved in the same biochemical reaction as an input or as two members of
1. Create mutations in Drosophila ID gene orthologs and analyze the same protein complex.
the effect on neural development and learning & memory.
2. Establishing functional interactions.
You can look at the wing of the fly to establish functional interactions. You can have targeted expression of the
gene or inactivation (shRNA). In transgene X there is an extra copy of MBD5 for instance or SHRNA against MBD5,
shRNA binds to messenger RNA and inhibits translation.
You have two stock lines, one is genomic enhancer of GAL4. This drives tissue specific expression of GAL4 and is
an enhancer for expression in the wing. If it drives expression you can get GAL4 (only in the wing).
UAS is a transcriptional activation of gene X. GAL4 binds to UAS and then it will drive the transcription of
transgene X.
Normal wing has no EHMT1 over expression. With overexpression you see deviations.
Then you double cross it and it has a knockdown in MLL3. If it is together with EHMT1
overexpression and knockdown of MLL3 then there is no wing. Thus the genes interact.
Because you have overexpression and knockdown of genes and you have a phenotype
that is clear that they work together. Agonistic means that
they work together in the same direction (whether good or bad) and antagonistic means they work against each
other. When they both have overexpression then they work together. An interaction between EHMT1 and MBD5
shows that the phenotype gets worse. The genetic defect is agonistic, they work together in the same pathway. So
EHMT1 module of interactions shows there are interactions between genes. There are also other
connection between disorders that have connections with EHMT1 modules like coffin-sirus syndrome.
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