Molecular Basis of Disease
Human molecular genetics
Lesson 0
Canonical example: CF
Most common monogenic disease.
Pancreas is blocked. Can’t get its stuff from
the pancreas to the intestines.
Immune reactive trypsinogen → needs to
go somewhere → ends up in the blood
Lung
Step 1: K+ in, Na+ out
Step 2: K+, 2 Cl-, and Na+ in
Step 3: CFTR → Cl- out
Step 4: Na+ and H20 follow → fluid mucus
Sweat test
1. Medication to sweat
2. Collect sweat
3. Test Cl- concentration
(sweat conductance)
4. High Cl-
concentration → CFTR
mutation → CF
1
,Skin
Skin CFTR → Cl- goes inside the cell
Normal sweat gland → same concentration Cl- in dermis and serum
CF sweat gland → chloride absorption is hindered by defective CFTR → higher Cl- concentration on
skin surface
Difference in Cl- transport lungs and sweat glands → lungs excretion, sweat glands absorption
CFTR
CFTR = cystic fibrosis transmembrane
conductance regulator
2
,CFTR variants
p.Phe508del is most
common mutation
Sequence variance →
variants that are
normal
DNA variants analyze
by exome sequencing
→ splice sites,
missense, nonsense,
frameshifts, in-frame
indel
Classification of CFTR mutations
1. Stop mutation
2. Protein gets
stuck in the Golgi
3. E.g., amino acids
block the channel
4. Missense
mutations, e.g.,
wrong folded
5. Nonsense
mutation
6. Protein is
transported back
to the Golgi
PTC → premature termination codon
(nonsense)
PTC read-through → stop is not
recognized
Orkambi
Ivacaftor → restores channel
function
Lumacaftor → facilitates folding
p.Phe508del → protein is not
folded correctly so it doesn’t leave
the Golgi. Lumacaftor makes sure
that it is folded correctly so the
protein will leave the Golgi and
Ivacaftor will give the channel a
boost to restore its function.
3
,SLC26A9 & CFTR
SLC26A9 → Cl- transport
Ivacaftor works less efficient
SLC = solute carrier family
Ivacaftor SLC26A9 variants
Intronic variants have effect on the efficiency of
ivacaftor.
Modifier gene variants:
TT → won’t get any extra volume (extra breath)
CC → will get extra volume
Ivacaftor does influence a modifier gene of CFTR.
Modifier genes
Modifier of phenotype genes.
Exocrine insufficiency →
pancreas
Lesson 1
Terminology
Variation → any deviation from the reference genome
Polymorphism → variation ≥1% of the alleles in a population
Mutation → variation <1% of the alleles in a population
Pathogenic → disease-causing mutation or polymorphism
4
,CNV → copy number variation → deletion or duplication ≥1 kb
Genome variation
Exome sequencing can find
SNVs and indels of <10 bp
because it only sequences 60
bp at a time.
CNVs are found by
microarrays or by whole
genome sequencing.
Variant nomenclature
Silent changes → c.6T>C; p.(=)
c = coding
number = position
p = protein
= = equal → between parentheses because of that it is assumed (protein is not sequenced, only the
DNA)
5
,Missense changes → c.4T>C; p.(Tyr2His)
Nonsense changes → c.10C>T; p.(Arg4*)
Frame shift deletion → c.12delA; p.(Met5fs*)
Insertion of duplication → c.12dupA;
p.(Met5Asnfs*)
In frame deletion/insertion → c.10_12delCGA;
p.(Arg4del)
Splice site changes → c.33-1G>A; p.(?) → -1 counts
for before the coding sequence
Fs leads to stop codon in the ~20 aa’s after the mutation
Acceptor splice site → before exon → AG (-1, -2)
Donor splice site → after exon → GT (+1, +2)
Annotate a promotor sequence → c.1-333C>T
Intellectual disability (ID) → Intelligence-quotient (IQ) <70,
limitations in adaptive functioning, present <18 yrs
Culturofamilial → multifactorial
Organic → something wrong with the brain →
small/big brain → monogenic
Kind of inheritance for ID → X-linked, autosomal recessive, de novo
ID: heterogeneous disorder
- Allelic heterogeneity
o Same/different phenotype
o Same gene
o Different mutations
- Locus heterogeneity
o Same phenotype
o Different genes
6
, Blood disease and ID.
Different types of mutations all over the
place. At first it was normal, but due to
the number of mutations the disease is
caused by different mutations.
Lots of ID is non-syndromic → ID but
doesn’t show.
Lots of genes are involved.
Hard to say which gene causes the
phenotype.
Genome-wide detection of variation
Lesson 2
<2009: first step karyotyping
Microscopic analysis → metaphase chromosomes →
condensed and thick enough to see through the
microscope → stain for banding pattern
7
,Del(16)(q13q22)
Deletion big
enough (1 band is
~5-10 million bp)
to see through the
microscope.
Guess for
breakpoints.
Karyotyping; limited resolution
- Genome-wide testing, but
o You can only see abnormalities when they involve
millions of nucleotides (much smaller aberrations can
cause aberrant phenotype as well)
o Resolution: ~500 bands, ~1 band per 6 million
nucleotides
o It is time-consuming, difficult to
automate, interpretation is subjective
Analysis chromosome indirectly to find very small
aberrations (submicroscopic) → has been done for
many years
- If the clinical features are specific, analysis of a
specific region (if you know where to look)
8
,FISH methodology
FISH = fluorescence in situ
hybridization
DNA really wants to be
double-stranded.
Heat DNA → denaturation
→ ss DNA → offer probe
directed to a part of the
chromosome that you know
→ probe fluorescently
labeled → check under
fluorescence microscope
Wolf Hirschhorn syndrome (deletion 4p)
FISH probe against Wolf
Hirschhorn region → 1
chromosome misses
this region → diagnosis
Genome diagnostics for ID +/- MCA (n=~2500 per year)
Genomic profiling with a microarray
- If you don’t know where to look based on the phenotype
- A microarray is a glass slide with a large quantity of (FISH)
probes
9
, How are arrays used
Isolate DNA from blood →
label DNA with green,
fluorescent dye
Reference DNA → label DNA
with red, fluorescent dye
→ hybridize DNA to slide with
probes → analyze signal
Green → duplication
Number of copies is difficult
to test with an array alone.
Red → deletion
Sensitivity and specificity
Female vs male
Test over reference log-ratio
Deletion → negative
Duplication → positive
More probes to the same region → all
up or down → change
Patient with a 3 Mb deletion in Prader Willi/Angelman syndrome region on chromosome 15
Prader Willi/Angelman → depends on if the
deletion is on maternal or paternal
chromosome
10
Human molecular genetics
Lesson 0
Canonical example: CF
Most common monogenic disease.
Pancreas is blocked. Can’t get its stuff from
the pancreas to the intestines.
Immune reactive trypsinogen → needs to
go somewhere → ends up in the blood
Lung
Step 1: K+ in, Na+ out
Step 2: K+, 2 Cl-, and Na+ in
Step 3: CFTR → Cl- out
Step 4: Na+ and H20 follow → fluid mucus
Sweat test
1. Medication to sweat
2. Collect sweat
3. Test Cl- concentration
(sweat conductance)
4. High Cl-
concentration → CFTR
mutation → CF
1
,Skin
Skin CFTR → Cl- goes inside the cell
Normal sweat gland → same concentration Cl- in dermis and serum
CF sweat gland → chloride absorption is hindered by defective CFTR → higher Cl- concentration on
skin surface
Difference in Cl- transport lungs and sweat glands → lungs excretion, sweat glands absorption
CFTR
CFTR = cystic fibrosis transmembrane
conductance regulator
2
,CFTR variants
p.Phe508del is most
common mutation
Sequence variance →
variants that are
normal
DNA variants analyze
by exome sequencing
→ splice sites,
missense, nonsense,
frameshifts, in-frame
indel
Classification of CFTR mutations
1. Stop mutation
2. Protein gets
stuck in the Golgi
3. E.g., amino acids
block the channel
4. Missense
mutations, e.g.,
wrong folded
5. Nonsense
mutation
6. Protein is
transported back
to the Golgi
PTC → premature termination codon
(nonsense)
PTC read-through → stop is not
recognized
Orkambi
Ivacaftor → restores channel
function
Lumacaftor → facilitates folding
p.Phe508del → protein is not
folded correctly so it doesn’t leave
the Golgi. Lumacaftor makes sure
that it is folded correctly so the
protein will leave the Golgi and
Ivacaftor will give the channel a
boost to restore its function.
3
,SLC26A9 & CFTR
SLC26A9 → Cl- transport
Ivacaftor works less efficient
SLC = solute carrier family
Ivacaftor SLC26A9 variants
Intronic variants have effect on the efficiency of
ivacaftor.
Modifier gene variants:
TT → won’t get any extra volume (extra breath)
CC → will get extra volume
Ivacaftor does influence a modifier gene of CFTR.
Modifier genes
Modifier of phenotype genes.
Exocrine insufficiency →
pancreas
Lesson 1
Terminology
Variation → any deviation from the reference genome
Polymorphism → variation ≥1% of the alleles in a population
Mutation → variation <1% of the alleles in a population
Pathogenic → disease-causing mutation or polymorphism
4
,CNV → copy number variation → deletion or duplication ≥1 kb
Genome variation
Exome sequencing can find
SNVs and indels of <10 bp
because it only sequences 60
bp at a time.
CNVs are found by
microarrays or by whole
genome sequencing.
Variant nomenclature
Silent changes → c.6T>C; p.(=)
c = coding
number = position
p = protein
= = equal → between parentheses because of that it is assumed (protein is not sequenced, only the
DNA)
5
,Missense changes → c.4T>C; p.(Tyr2His)
Nonsense changes → c.10C>T; p.(Arg4*)
Frame shift deletion → c.12delA; p.(Met5fs*)
Insertion of duplication → c.12dupA;
p.(Met5Asnfs*)
In frame deletion/insertion → c.10_12delCGA;
p.(Arg4del)
Splice site changes → c.33-1G>A; p.(?) → -1 counts
for before the coding sequence
Fs leads to stop codon in the ~20 aa’s after the mutation
Acceptor splice site → before exon → AG (-1, -2)
Donor splice site → after exon → GT (+1, +2)
Annotate a promotor sequence → c.1-333C>T
Intellectual disability (ID) → Intelligence-quotient (IQ) <70,
limitations in adaptive functioning, present <18 yrs
Culturofamilial → multifactorial
Organic → something wrong with the brain →
small/big brain → monogenic
Kind of inheritance for ID → X-linked, autosomal recessive, de novo
ID: heterogeneous disorder
- Allelic heterogeneity
o Same/different phenotype
o Same gene
o Different mutations
- Locus heterogeneity
o Same phenotype
o Different genes
6
, Blood disease and ID.
Different types of mutations all over the
place. At first it was normal, but due to
the number of mutations the disease is
caused by different mutations.
Lots of ID is non-syndromic → ID but
doesn’t show.
Lots of genes are involved.
Hard to say which gene causes the
phenotype.
Genome-wide detection of variation
Lesson 2
<2009: first step karyotyping
Microscopic analysis → metaphase chromosomes →
condensed and thick enough to see through the
microscope → stain for banding pattern
7
,Del(16)(q13q22)
Deletion big
enough (1 band is
~5-10 million bp)
to see through the
microscope.
Guess for
breakpoints.
Karyotyping; limited resolution
- Genome-wide testing, but
o You can only see abnormalities when they involve
millions of nucleotides (much smaller aberrations can
cause aberrant phenotype as well)
o Resolution: ~500 bands, ~1 band per 6 million
nucleotides
o It is time-consuming, difficult to
automate, interpretation is subjective
Analysis chromosome indirectly to find very small
aberrations (submicroscopic) → has been done for
many years
- If the clinical features are specific, analysis of a
specific region (if you know where to look)
8
,FISH methodology
FISH = fluorescence in situ
hybridization
DNA really wants to be
double-stranded.
Heat DNA → denaturation
→ ss DNA → offer probe
directed to a part of the
chromosome that you know
→ probe fluorescently
labeled → check under
fluorescence microscope
Wolf Hirschhorn syndrome (deletion 4p)
FISH probe against Wolf
Hirschhorn region → 1
chromosome misses
this region → diagnosis
Genome diagnostics for ID +/- MCA (n=~2500 per year)
Genomic profiling with a microarray
- If you don’t know where to look based on the phenotype
- A microarray is a glass slide with a large quantity of (FISH)
probes
9
, How are arrays used
Isolate DNA from blood →
label DNA with green,
fluorescent dye
Reference DNA → label DNA
with red, fluorescent dye
→ hybridize DNA to slide with
probes → analyze signal
Green → duplication
Number of copies is difficult
to test with an array alone.
Red → deletion
Sensitivity and specificity
Female vs male
Test over reference log-ratio
Deletion → negative
Duplication → positive
More probes to the same region → all
up or down → change
Patient with a 3 Mb deletion in Prader Willi/Angelman syndrome region on chromosome 15
Prader Willi/Angelman → depends on if the
deletion is on maternal or paternal
chromosome
10
