Semester 1 Comprehensive

USMLE Review
Atheer R. Hussain-Amin, B.Sc.
M.D. Candidate
 1. Chromatin Structure
• DNA exist in the condensed form called
chromatin to fit the nucleus.
• Negatively charged DNA loops twice
around positively charged histone
octamers forming a nucleosome (DNA
+ Histone).
• Histones are rich in lysine and arginine.
• H1 Histone binds to the linker DNA
and stabilizing the chromatin fiber.
• In mitosis, DNA condenses to form
chromosomes.
• DNA and Histones synthesis occurs in
S phase of the cell cycle.
• Nucleosome core = 2 x [H2A, H2B, H3,
and H4]
 Chromatin Structure
• Heterochromatin: Condensed, transcriptionally
inactive, and sterically inaccessible
• Euchromatin: less condensed, transcriptionally
active, and sterically accessible
• DNA Methylation: does NOT change base pairing
properties
•
• Eukaryotes methylate only C while prokaryotes
methylate both A and C.
• C’s followed by a G can be methylated as in the
CpG islands.
• A’s in a GATC sequence could be methylated only.
• DAM (DNA Adenine Methylase) in bacteria
• DNMT (DNA Methyl Transferase) in eukaryotes
• It occurs in DNA (template strand) replication
allowing for mismatch repair enzymes distinguish
the old strand from the new strand. However, if
methylation occurs in CpG Islands it represses txn.
• Histone Methylation: reversible DNA txn repressor
but could be an activator in rare occasions.
• Histone Acetylation: txn activator because it
relaxes DNA coiling.
2. Nucleotides
• Purines: AG
• Pyrimidines: CUT and 1 ring
• Deamination of C results in a U
• Deamination of 5-Me-C results
in T
• G-C has 3 H bonds and
increasing it in DNA results in
a larger Tm for DNA.

Induce these rxns

 3. Genetic Code

• 1 codon=1 AA
• 1 AA= multiple codons
• Except: Methionine= AUG and Tryptophan= UGG
• Read the transcript from a specific starting point as a continuous sequence of bases
except some viruses.
• The genetic code is conserved and universal in humans except in Mitochondria where
the code is different.
Codon "Universal code" human mitochondria
UGA STOP Trp
AUA Ile Met
AGA
Arg STOP
AGG
4. DNA Replication
Proteins required for Replication

Prokaryotes Eukaryotes Function

DnaA ORC proteins ORI recognition

Gyrase Topoisomerase II Removal positive supercoils

DnaB Mcm Helicase
DnaC Cdc6 Helicase loader
SSB RPA Maintains/protects ssDNA
γ-complex RFC Clamp loader
DNA Pol III DNA Pol δ Primary replicating enzyme
β-subunit PCNA Clamp
Primase Primase RNA primer synthesis
Pol α Extends RNA primer for 10-20 nt

DNA ligase DNA ligase Links adjacent Okazaki fragments

DNA Pol I FEN-1/RNaseH Remove RNA primer
DNA Pol I DNA Pol ε Fills gap left by primer

This is Ori in prokaryotes

DNA Replication

• Delay in Methylation
@ E. Coli Ori ensures
orderly DNA
replication.
• DNA Ligase
mechanism of action.
• Gyrase resolves the
positive supercoiling
that occurs in front of
the replication fork. DNA Pol III DNA Pol I

5'-3' Polymerase Yes Yes

3'-5' Exonuclease Yes Yes
5'-3' Exonuclease No Yes
Polymerisation rate high low
Processivity high low
DNA Replication in Prokaryotes
DNA Replication (Telomerase)

• It synthesizes DNA in an
RNA dependent manner.
• 5’ to 3’ syntheses starting
at the 3’ end of the
chromosome.
• It is encoded by the TERC
gene
5. Mutation in DNA
• Severity of the damage:
• Silent<<missense<nonsense<frameshift
• For the first three types of mutation:
• Transition mutation: it is a purine to a purine OR a pyramidine to a pyramidine type of
mutation.
• Transversion mutation: it is a purine to a pyrimidine OR a pyramidine to a purine mutation
• Silent: same AA it is a result of the 3rd codon being changed (tRNA
wobble chemical property)
• Missense: one nucleotide substitution resulting in a changed AA
• Conservative missense mutation is when the alternative AA has similar physio-
chemical properties
• Results in sickle cell disease
• Nonsense: early stop codon
• Frameshift: deletion or insertion that is NOT a multiple of 3s. It results in
a truncated or non-function protein.
• Such as Duchenne Muscular Dystrophy.
Mutation in DNA
Frameshift mutation in COL2A1 gene resulting in
premature termination codon (PTC). PTCs do not result in
truncated polypeptides

(EJCs)

Ribosomes stall at termination codons

Nonsense-mediated mRNA decay (NMD) machinery investigates
If there are EJCs downstream then it is a PTC - because real stop
codons are almost always in the last exon - and the mRNA will be
degraded
If no downstream EJCs then it is the real stop codon and the
ribosome falls off to reinitiate on this or another transcript
6. DNA Repair
• Nucleotide Excision Repair (NER)

• DNA Helicase= XPB and XPD

• The endonuclease involved is called exinuclease.
• It involved in repairing bulcky helix distorting damages
such as a pyramidine dimer OR nucleotides with
chemical groups attached to them.
DNA Repair
• Xeroderma pigmentosum: AR disease. It is a rare
photosensitivity with an increased risk of malignant
melanoma upon sun exposure.
• Can be tested in the lab via measuring new DNA synthesis
after cells have been exposed to UV-light: blood lymphocytes
of XP patients are unable to synthesise new DNA after UV
exposure.
• 7 genes for XP-A to XP-G. XP-C is the most common and milde
form. All genes have a role in NER.
• Cockayne Syndrome: is a disease of the transcription-
coupled NER. Autosomal recessive, rare, photo
sensitivity, arrested growth, neurological disorder, but
no increased cancer risk. Genes: CSA (and CSB)
 DNA Repair
• Base Excision Repair: it involves
a base specific glycosylase which
will create an AP site
(apurinic/apyrimidinic).
• AP endonuclease removes a one
or more nucleotide. This
enzyme cleaves the 5’ end while
lyase cleaves the 3’ end.
• DNA polymerase is DNA pol β.
• It is involved inrepairing
spontaneous/toxic-induced
deaminations.
DNA Repair
• Mismatch Repair: a defect
in this repair machinery
results in HNPCC=
Hereditary nonpolyposis
colorectal cancer
DNA Repair
• Nonhomologous End Joining
Repair (NHEJR): this repair
machinery brings together 2
DNA ends repairing a double
stranded break. Does not
require homology.
• A mutation in this machinery
results in Ataxia Telangiectasia it
is AR inheritance. This disease is
due to a defect in ATM gene
located in 11q22.3. ATM is a
serine/threonine protein kinase
required in NHEJR.
• The onset is in toddler years.
The sclera will have
Telangiectasia by the age of 5 to
8.
• It results in neurodegeneration
disease
7. Directions of Synthesis
• DNA and RNA both synthesized from 5’ to 3’ end

• Proteins synthesized N to C-terminus.

• mRNA is read 5’ to 3’
• Drugs designed to block DNA synthesis are designed to have
a 3’-OH that prevent further nucleotide addition to the
growing chain
Important Codes and Organizations
• AUG =start
• In rare occasions it will be GUG but VERY RARE
• In euckaryotes AUG codes for methionine. It is
usually removed before the completion of trn.
• In prokaryotes it codes for f-met
• UGA, UAG, and UAA = Stop

5’

Intron
AATAAA
Exon
TATAAT

Exon
Enhancer ATATTA TTATTT
3’
Template/antisense Promoter/TATA Box Termination Signal
/non-coding strand for poly A tail
+1 (start of txn)
8. Gene Expression
• Promoter: site where RNA polymerase and multiple
other txn factors bind to DNA upstream from gene
locus (AT-rich upstream sequence with TATA and
CAAT boxes).
• Mutation at this site decrease gene expression
• Enhancer: a DNA segment that alters DNA
expression via bining TFs that will interact with the
gene expression machinary
• Silencer: repressor binding site
• both enhancers and repressor could be locate anywhere
even inside the gene (the introns).
 9. RNA Polymerase
Eukaryotic RNA polymerases
• I is numerous, II is
massive, III is tiny. Effects of alpha-
• They do not have amanitin (from
Type Location Cellular transcripts
a proofreading mushrooms Amanita
mechanism but phalloides)
could initiate
synthesis without 18S, 5.8S, and 28S
a primer. I Nucleolus Insensitive
rRNA
• RNAP II opens
DNA at promoter
regions. mRNA precursors
II Nucleoplasm Strongly inhibited
• Death cap snRNA and miRNA
mushrooms
cause tRNA Inhibited by high
hepatotoxicity III Nucleoplasm
and 5S rRNA concentrations
10. Eukaryotic RNA processing
• The RNA found in the nucleus is
called hnRNA which will then
become mRNA.
• Poly(A) tail is about 200 A’s
• Splicing out introns.
• Poly A is added independently
via poly A polymerase enzyme
that recognizes AAUAAA
sequence and does NOT require
a template.
• mRNA quality control occurs in
the cytoplasmic processing
bodies (P-Bodies) that contain
exonuclease, decapping proteins,
and miRNA. Some mRNA may be
stored in these bodies if it
 11. Splicing of pre-mRNA (hnRNA)
- snRNPs are involved
specifically to form the
splicing machinery
(splicesome)
- Lariate-Shaped components
are involved in an
intermediate

Antibodies to splicesomal snRNPs (called anti-smith

antibodies) are highly specific to SLE (systemic
lupus erythematosus) where 90% of cases occur in
women and it is a chronic inflammatory disease.
Anti-U1 RNP is associated with mixed connective
tissue disease.
Introns and Exons
• Exons code for genetic information in the transcript for
hnRNA (codes for mRNA), pre-tRNA (tRNA), pre-rRNA
also known as 45S (18S, 5.8S, 28S rRNA).
• rRNA does NOT have a poly(A) but does have a cap. tRNA
does not have a cap or a poly(A). miRNA has bth cap and
poly(A).
• Different exons of a gene are spliced together in
different environments or cells-Alternative splicing. This
allows to produce a greater variety of proteins.
• Abnormal splicing results in abnormal oncogenesis and
blood disorders such as betta-thalesemia.
• Introns= intervening and do not code anything. They
stay in the nucleus.
12. tRNA-Structure
• CCA= at the 3’ aminoacyl end it is called the acceptor stem.
• T-Arm (near the 3’ end): contain TΨC (thymine, pseudouridine,
and cytosine). This sequence is used to bind to the ribosome.
• D-Arm (near the 5’ end): contains dihydrouracil residues
important for the aminoacyl-tRNA synthetase to recognize.
• Variable arm is found near the T-arm.
tRNA-Charging
• 1 Aminoacyl-tRNA synthetase
per 1 AA. It uses one ATP
molecules but TWO energy
equivalents.
• First it binds to the AA then to
the tRNA. If the two molecules
are incorrectly joined the bound
is hydrolyzed. The energy in the
AA-tRNA is enormous and drives
the peptide bond formation.
• Accuracy of protein synthesis in
an Amino Acid level is done by
Aminoacyl-tRNA synthetase +
binding of the charged tRNA to
the correct codon on mRNA.
• mRNA needs to be accurate in
the first 2 nucleotides of the
codon but the 3rd could be. The
third is the wobble position.
 ONLY mRNA with an ORF(Open Reading Frame) codes for proteins

13. Protein Synthesis(translation)-

Initiation

These processes occur in

prokaryotes and GTP
hydrolysis initiate
translation. IFs help form
the assemble.
 Peptidyl Transferase

Protein Synthesis-Elongation activity is found in

the large subunit
Protein Synthesis-Termination
Prokaryotic Drugs:
• Streptomycin: binds to 30S subunit
and causes misreading of the
genetic code and inhibits initiation of
translation at higher concentrations
• Puromycin: structurally similar to
the 3’end of aminoacyl tRNA: binds
to the A site and participates in
peptide bonding: peptidyl-puromycin
(premature ending of synthesis)
• Tetracyclin: blocks A-site
• Chloramphenicol: binds to the
large ribosomal subunit and blocks
bacterial peptidyl transferase
• Erythromycin: binds also the large
ribosomal subunit but translocation
is now blocked
Eukaryotic translation

Diphtheria toxin: inactivates eukaryotic

elongation factor 2 (eEF2, similar to bacterial
EF-G) by modification of a histidine residue
(ADP –ribosylation). Diphtheria is an upper
respiratory track illness that sometimes can
cause neck swelling.
Ricin: toxic protein from castor beans:
depurinates a specific adenosine residue in
the 28S rRNA, thereby inactivating its
peptidyl transferase activity
NOTE: eEF1 is orthologous to EF-Tu
 14. Posttranslational Modification
• Trimming: removes either N- or C-terminals
• Covalent alterations: phosphorylation, ubiquitination,
glycosylation…etc
• Chaperon Proteins: These are protein machines that maintain and
facilitate protein correct folding. They have affinity to hydrophobic
protein portions
• Heat shock chaperons in yeast prevent protein folding disruption
incase of increased temperatures.

Co-translationally
Could be found in
mitochondria. In addition,
to cytoplasm.
 15. Cell Cycle phases
• Checkpoints control transitions between phases of the cell
cycle.
• Cyclins and CDKs + tumor suppressors both regulate the checkpoints
of transition.
• CDKs are constitutively expressed in an inactive form.
• Cyclins are expressed in specific phases of the cell cycle abd
activate CDKs
• Cyclin-CDK complex are activated and inactivated in
synchrony for the cell to go through the cell cycle.
• Tumor suppressors such as TPp53 and hypophosphoryated Rb
normally inhibit G1-to-S progression
• Li-Fraumeni syndrome (rare form of hereditary cancer)
• SBLA cancer= Sarcoma, breast, leukemia, adrenal gland
• Causes multiple malignancies at early age
• Cell Types:
• Permanent: Remain in Go and they regenerate from stem cells.
Hypophosphorylated
(Neurons, skeletal, cardiac, and RBCs) Rb and p53
• Stable (quiescent): Enter G1 from Go upon stimulation.
(Hepatocytes and Lumphocytes)
• Labile: Never goes to Go, divided rapidly with a short G1. They are
affected severely by chemotherapy. (bone marrow, gut epithelium,
skin, hair follicles, and germ cells).
 16. Endoplasmic Reticulum
• RER (goblet cells and plasma cells)
• It is site of synthesis for secretory
proteins and ALL TRANSMEMBRANE
proteins
• N-linked oligosaccharides
• Nissle bodies (only in neurons) is
the site of synthesis of peptide NTs
• Free Ribosomes-site of synthesis of
organelle and cytosolic proteins
• SER (hepatocytes, adrenal cortex
(steroid producing cells), and
gonads)
• Steroid synthesis and detoxification
of drugs/toxins.
• It also contains Glycogen
degredation and gluconeogenesis
• Because it contains G-6-Pase
 Cystic Fibrosis

• Autosomal recessive

• Mutation in CFTR on chrom7

: Cl- ion channel
(plasma membrane protein across
epithelial membranes)

• Predominant mutation (ΔF508)

→ incorrectly folding protein
• Most common lethal disease in
Caucasians
• Would function normally if
transported to plasma membrane

• But protein = ejected from ER back

to cytosol & degraded by proteasome
→ Cystic fibrosis
 Familial Hypercholestermeia

Results in elevated LDL levels due to a defect in the LDL receptor.

Results in: Atherosclerosis early on life, corneal arcus, tendon xanthomas especially in the
Achilles tendon
 Cholesterol uptake
•Cholesterol transported in blood as cholesteryl esters : LDL
(LDL=lipid-protein particles)

•Cholesterol needed for membrane synthesis

→ expression LDL-R on surface (diffuse to clathrin pits)

•Endocytosis

•Vesicle fusion with endosome

Low pH
LDL dissociate from LDL-R
LDL to lysosome:
cholesterol esters in LDL
hydrolysed to free cholesterol
 17. Cell Trafficking-Overview
Direct protein to a particular cellular location….

• Default→ cytosol
• NLS → nucleus
• SKL → peroxisomes
• Mitochondrial
signal sequences

• ER signal sequence
→ rER
• KDEL → ER (soluble)
• KKXX → ER (insoluble)
• M6P → lysosome
• Default:
→ plasma membrane
→ secretion
Cell Trafficking-Golgi
- It is the distribution center for proteins and lipids.
- Modifies N-oligosaccharides
- Adds O-oligosaccharides on S and T
- Adds the Mannose-6-Phosphate (M6P) signal on the
N-oligosaccharides added in the ER.
- COP I= golgi golgi (retrograde); golgi ER
- COP II= golgi golgi (anterograde); ER Golgi
- Clathrin= TGN Lysosomes; PM endosomes
(receptor mediated endocytosis

M6P signal synthesis in the Golgi

 Vesicle Coat Assembly
• ARF & SAR = G proteins (GTPases)
• ARF: COP I (& Clathrin), SAR: COP II
• GEF recruits ARF/SAR, GDP → GTP
• ARF/SAR promotes coat protein assembly
• GTP → GDP: ARF/SAR disassociates from membrane, COP coat
disassembles
 Vesicle fusion (neural)

• SNARE proteins provide specificity

• v (vesicle)-SNARE (synaptobrevin) proteins interact
with
t (target)-SNARE (syntaxin) proteins, to form complex
It interacts with calcium sensor called synaptotagmin
• RAB-GTP regulates initial docking & binding of SNAREs.
GTP then hydrolyzed to GDP

• NSF/SNAPs binds to complex & induce membrane

fusion
Vesicle Fusion-Clinical Significance
• Botulism: is due to the botulin
toxin from Clostridium
botulinum found in not
sterilized canned food. The
neurotoxin cleave
synaptobrevin where th Ach
cnnot be release to the NMJ.
• This results in flaccid paralysis
especially the respiratory and
skeletal muscles.
• Tetanus: is due to Clostridium
tetani that is found in
contaminated wounds. It
prevents fusion of GABA and
glycines (inhibitory NTs) from
motor neurons.
• Spastic Paralysis
 Endocytosis
• Pinocytosis
• This is constitutive and involves
actin remodeling of the
cytoskeleton.
• Phagocytosis
• Opsonins: these are proteins that
are recognized by the phagocyte
and ingest material in a receptor
mediated and clathrin-independent
manner.
• When it digest the material it will
either form a residual body that if
accumulated will be lipofucins or
excreted
• Clathrin Mediated Endocytosis
(Receptor Mediated)
• Adaptins: these molecules control
coated vesicle formation, where
they selectively target appropriate
cargo receptors. They also
accumulate different types of
receptors
• Caveolae Coated Vesicles is spiked
Receptor-mediated endocytosis
 Cell Trafficking-Endosomes
• Late endosomes contain
material from endocytosis
& hydrolytic enzymes
• Become lysosome when
most material degraded.
Can reenter the system by
fusing with another late
endosome
• Primary lysosomes
• Bud from TGN, contain
newly synthesized acid
hydrolases
• Not yet acquired material to
be digested

• Secondary lysosomes
• Fusion of primary lysosome - They are sorting centers for material from outside
with substrate to be
degraded of the cell + Golgi
• In various stages of - Then they send the material to lysosomes for
degradation
destruction or back to the PM/Golgi for further
usage (recycling)
Cell Trafficking-Endosome
Maturation
• Maturation of early endosomes
containing endocytic vesicles to
late endosomes occurs via
“Multivesicular bodies”

• MVBs shed vesicles to recycle

material back to PM
• Gradually convert into late
endosomes by fusing with
eachother / with other late
endosomes

• Late endosomes become

endolysosmes & lysosomes by
i. Fusing with preexisting
lysosomes
ii. Progressive acidification
Cell Trafficking-Lysosomes and hydrolases

• Lysosomes: digest material via acid hydrolases in an acidic environment of

pH= 4.5. The inner membrane of the lysosome contains glycocalyx coat to
protect itself.
• V-type ATPase in the membrane achieves the acidic environment via pumping
H+ into the lysosome.
• The acidic conditions detach the cargo from their receptors.
• The cargo’s phosphates are removed so that they cannot re-bind to the M6P receptor.

Lysosomal
Hydrolase
precursor
Early endosome
18. Lysosomal Storage Diseases-I-cell disease (Mucolipidosis II)
• Inclusion cell disease: this is an inherited lysosome storage
disorder.

• Deficiency of N-acetylglucosamine
phosphotransferase
→ Absense of M6P tag

• Acid hydrolases lacking M6P are

secreted extracellularly
→ Waste products accumulate as inclusion bodies

• Skeletal abnormalities (lack of growth)

• Coarse facial features
• Clouded cornea
• Restricted joint movement
• High plasma concentration of lysosomal enzymes
• Psychomotor retardation
• Enlarged liver, spleen, heart valves
• Death CHF / RTI
• Life expectancy <10yrs (fatal)
 Pseudo-Hurler Polydystrophy
(Mucolipidosis III)

• Milder form of I-cell disease

• Later onset
• Survival into adulthood
 Lysosomal storage diseases

Nuclear membrane

Ganglion in Tay Sachs Gaucher cell

Prominent lyosomes with whorled configuration Elongated distended lysosomes
 Lysosomal storage diseases
• Mucopolysaccharidoses (accumulation of sulfated polysaccharides / GAGs)
• Hunter syndrome
• Hurler syndrome
• Sanfilippo syndrome
• Morquio syndrome
• Maroteaux-Lamy syndrome
• Sly syndrome

• Lipid storage disorders / Sphingolipidoses (accumulation of sphingolipid)

• Gaucher's disease (accumulation of glucocerebroside)
• Niemann-Pick disease (accumulation of sphingomyelin & cholesterol)
• Gangliosidoses; Tay Sachs disease (GM2 gangliosidosis)

• Mucolipidoses (accumulation glycoprotein & glycolipid)

• I : sialidosis
• II: I-cell disease
• III: pseudo-Hurler polydystrophy

• Leukodystrophies
 Degradation of
sphingolipids
&
Sphingolipidoses
 Mucopolysaccharidoses
Defective degradation of GAGs (mucopolysaccharides)
MPS I – MPS VII
All autosomal recessive except Hunter (X-linked)

• Coarse facial features

• Corneal clouding in some
• Joint stiffness & Skeletal deformity
• Hepatosplenomegaly
• Mental retardation (Lesions in the brain)
• Hirsutism in some
• Arterial depostits (coronary arteries)
• Urinary excretion of the accumulated
GAG often increased
 Hurler syndrome (MPS IH)
• Most severe MPS, (MPS I)
• Deficiency of α-L-iduronidase
→ accumulation of dermatan sulphate & heparan sulphate

• Initially normal growth, but…

• At few months old:
• Physical & mental deterioration
• Growth stops at 2-4 yrs
• Hepatosplenomegaly
• Deafness
• Skeletal deformity
• Coarse facial features
• Hirsutism
• Thickened skin
• Corneal clouding

• Death ≤10 yrs

Hurler syndrome (MPS IH)
 Scheie & Hurler-Scheie syndrome
(MPS IS & MPS IHS)
• Residual α-L-iduronidase activity
• Milder disease (Scheie = mildest MPS I)

MPS IS

MPS IHS
 Hunter syndrome (MPS II)

• Deficiency of iduronodate sulphatase (X-linked)

→ accumulation of dermatan sulphate & heparan sulphate

• Similar to Hurler syndrome BUT

• Later presentation (2-4 yrs) &
milder course (survival into 30’s)
• no corneal clouding
• X-linked, not autosomal recessive
 Sanfilippo syndrome (MPS III)
• Defect in heparan sulphate degradation (types A-D)

• Normal development for first 1-2 yrs, followed by

• Progressive mental retardation & increasing behavioural disturbance
• Aggressive behaviour & destructiveness
• Hyperactivity
• Sleep disturbance
• Hearing loss
• Progressive immobility, dysphagia, seizures & dementia
• Mild facial dysmorphism (no hirsurtism)
• Death late teens/early 20’s

(Hurler = most severe MPS but children with Sanfilippo live longer with more
severe behavioural problems)
 Morquio syndrome (MPS IV)

• Defective degradation of keratan sulphate

• Deficiency of:
A. Galactosamine-6-sulphatase (MPS IV A)
B. β-galactosidase (milder) (MPS IV B)

• Short stature
• Kyphoscoliosis
• Pectus carinatum (pigeon chest)
• Deafness
• Weakness
• Aortic regurgitation → cardiomegaly
• Normal IQ

• Often considered skeletal dysplasia rather than storage

disease
 Maroteaux-Lamy syndrome (MPS VI)

• Deficiency of arylsulphatase B
• Similar to Hurler but normal IQ

Corneal opacification

Sly syndrome (MPS VII)

• Deficiency of β -glucoronidase
• Wide variability in severity; different mutations
19. Cotranslational Translocation
• The ER signaling sequence is usually in the N-terminal.
• SRP is a cytosolic ribonucleoprotein.
 Cotranslation Translocation
• BiP (Binding Protein): lumenal
ER chaperone (help proteins
fold), binds peptide in ER
lumen & pulls it in
• Signal peptidase cleaves off
N-terminal signal peptide as
protein enters ER lumen
• Some transmembrane
proteins lack cleavage site &
retain signal peptide
sequence (internal signal
peptides)
20. Peroxsiomes
• These are membrane enclosed organelles
involved in VLCFA metabolism (24 or more
C’s) when it reaches 10C’s the job will be
done in mitochondria, amino acid
metabolism and branched chain fatty acids.
• pH= 7.5 with the enzymes made on free
ribosomes
• Present in all tissues
• (liver & kidney)

• Carry out oxidative reactions

• Generate H2O2 for oxidative purposes

• Contain catalase (peroxidase) to destroy XS

H2 O2

• Involved in biosynthetic, degradative &

detoxification reactions
• PEX =the gene for peroxin
• It synthesizes plasmogens (ether
phospholipids, bile, cholesterol, and
dolichol which is also made in sER.
 Peroxisome: Synthetic function

• Plasmalogen synthesis
• Phospholipids with HC chain linked to
glycerol by ether bond (not ester bond)
• Membrane components of heart & brain
(80-90% of myelin membrane phospholipid)
• Deficiency → abnormalities in nerve cell
myelination

• Bile acid synthesis

• Lipid biosynthesis
• Cholesterol & dolichol (most made in sER)
Peroxsimes-Clinically Oriented
• Involved in gout via purine degradation where
hyperuricaemia is the cause. Thus, use Allopurinol
to inhibit Xanthine oxidase.
 Peroxsimes-Clinically Oriented
Zwellweger Syndrome
• Autosomal recessive, congenital
• Peroxins don’t recognise Ser-Lys-Leu (SKL)
• → Failure to import peroxisomal enzymes
• Peroxisome deficiency
• VLCFA accumulates in blood & tissues
• Lack of plasmalogen

• Accumulation of VLCFAs in glial cell membrane

• (no β-oxidation) → abnormal brain development
• neuronal migration defects
• hypomyelination

• Accumulation of VLCFAs in liver → hepatomegaly & liver failure

• Lack of bile acids → ↓ fat absorption → ↓ ATP → muscle

weakness
• Severe neurological dysfunction
• Hypotonia
• Hyporeflexia
• Seizures
• Mental retardation
• Dysphagia

• Dysmorphic features
• Prominent forehead
• Hypertelorism
• large fontanelles

• Hepatomegaly & liver dysfunction

• Death 6-12 months

Peroxismes-Clinically Oriented
X-linked Adrenoleukodystrophy (XALD)
• Most common peroxisomal disorder

• Defect in transport of VLCFA into peroxisome

• → defective breakdown of VLCFAs

• VLCFAs accumulate in
• Brain (glial cells) → myelin breakdown
• Adrenal cortex → adrenal atrophy

• Onset 5-10yo: apathy, behavioural changes

• Spasticity, ataxia, visual loss
• Death few years later
21. Proteasomes
• If a protein cannot be fixed with
chaperones, it is:

1. Ejected back through the

translocon to cytosol
2. Deglycosylated & Ubiquitylated
• Cytosolic ubiquitin attached
to misfolded protein; poly-
ubiquination
It has been shown that this system has
3. Complex taken up by proteasome,
degraded to amino acids a contributing factor in Parkinson’s
disease

• Proteosomal degradation = Non-

Lysosomal protein degradation
22. Microtubule
• Each dimmer has the capacity
to bind to 2 GTPs.
• Found in= Flagella, Cilia, and
mitotic spindle.
• Grows slowly but collapses
quickly.
• Involved in slow axoplasmic
transport in neurons.
• KADR= Kinesine is Anterograd
and Dynein is Retrograd.
• GTP-bound β-tubulin: α/β
dimer has high affinity for
other subunits
• GDP-bound β-tubulin: α/β
dimer has lower affinity
Microtubule
• dimers = added / subtracted from the
hollow tube

• Dimers add more easily to the (+)

end, especially before GTP hydrolysis

• Growth rate = proportional to

concentration of GTP-tubulin dimers

• GTP region = growth cap

• β-tubulin hydrolyses GTP→GDP

• If hydrolysis catches up with dimer

addition, MT will shrink =
“catastrophe”

• γ-tubulin = scaffold for growth in the

microtubule organising centres (MTOCs);
centrosome, basal body
Microtubule
• MAP= Microtubule
Associated Proteins
• tau (MAP) →
hyperphosphorylated
tau
• Forms NFTs
• ↓ functional MT
(depolymerisation
& disruption of
axon transport)
• To eventually
cause Alzehimer’s
Disease
 Alzheimer's Disease

• β-amyloid / senile plaques

(extracellular)
• accumulation of β-amyloid
peptide (β-A42/43) , apoE also
present→ progressive neuronal
damage
• Familial, autosomal dominant (5-10%) - Early
onset (<60): Presenile
• Presinilin 1
• Presinilin 2
• β-amyloid precursor
• (trisomy 21)

• Predisposition - Late onset (>60): Senile

• Apolipoprotein E (ε4 allele)
• Late onset = Multifactorial
• Apo E4 is a major risk factor
• Apo E2 reduces the risk
 Chédiak-Higashi syndrome-an immune system disease

• Rare, autosomal recessive

• Mutation CHS1/LYST = lysosomal trafficking regulatory protein
normally involved in vesicle fusion
• Delayed fusion of phagosome with lysosome in leucocytes
• Autophagocytosis of melanosomes in melanocytes → albinism
• Granular defects in NK cells & platelets

• Recurrent infections (life threatening)

• Hypopigmentation
• Mild coagulation defects
• Varying neurological problems
• Treatment = BMT (haematologic & immune defects)

BUT….
• USMLE = defect in microtubule polymerisation that causes defects in
cytoplasmic granules
Microtubule-Drugs
• BIND TUBULIN SUBUNITS & PREVENT POLYMERISATION
• Colchicine
• Used to treat gout

• Vincristine, Vinblastine
• Treatment of cancers with high mitotic index

• Mebendazole (anti-helminthiea)
• Griseofulvin (anti-fungal)

• BIND & STABILISE MTs (inhibit depolymerisation)

• Paclitaxel (Taxol)
• Blocks mitosis (can’t break down mitotic spindle)
• Treatment of cancers
23. Cillia
• Axonemal Dynein is an ATPase.
CXR
 Primary Ciliary Dyskinesia (PCD)
• Autosomal recessive, Variable penetrance
• 1 in 15,000 - 40,000 births

• immotile cilia & sperm

→ retention of secretions &
recurrent infection (bronchitis,
otitis media & sinusitus)
→ infertility

• 50% = Kartagener syndrome;

• Bronchiectasis
• Situs inversus (Dextrocardia)
• Chronic paranasal sinusitis
• Infertility
• Ectopic pregnancy
PCD / Kartagener syndrome

↓ ciliary activity affects

cell motility during
embryogenesis
→ situs inversus
 PCD / Kartagener syndrome

• Total lack of dynein arms or

• Defect of inner dynein arms

→ immotility of cilia & sperm

24. Actin-Overview
• Actin (G-Actin polymerizes to F-actin) and myosin II
(dimeric, ATP-driven motor protein): responsible for
muscle contraction, cytokinesis, Adherin junctions.
• Cell surface shape & Cell motility
• Cell division & cytokinesis
• Lamellipodia, filipodia, phagocytic cups
• Microvilli (intestinal epithelial cell surface) & stereocilia
• Cell junctions

• Muscle contraction

• vesicle & organelle movement

• Form contractile stress fibers that have the capacity to form tubes
such as the embryonic neurotube.
• Forms the cleavage furrow
Actin-polymerization

• High concentration of ATP bound G-actin

• → monomers add to both ends, faster at (+) end

• Lower concentration of ATP bound G-actin

• → monomers add to (+) end & come off (-) end
• = treadmilling : length of the filament remains the same
Actin-Nucleation
• Nucleation occurs at plasma membrane

• Highest density of actin filaments at cell periphery

• Cell cortex (beneath plasma membrane) = actin filaments;
→ determines cell shape / motility

• External signals regulate nucleation of actin filaments at plasma membrane: rapid changes
in shape & stiffness in response to environment
• Arp2/3 (actin-related proteins) stimulates nucleation of G-actin into dimers/trimers (initiates
polymerisation). It nucleates/binds preexisting filaments more efficiently. Thus, forming
branched network.
• It is important in the leading edge of cell migration
• G-actins bind together into dimers / trimers (a nucleus)
• Elongation begins: actin monomers added to nucleus or existing filament with higher affinity;
ultimately form double helix (F-actin)
• Monomers need bound ATP to add to filament / nucleus
• Profilin exchanges ADP for ATP, readying the monomer for attachment
• Formins nucleate growth of straight unbranched actin filaments
• cross-linked by other proteins → parallel bundles.
1) Cross-linking proteins (filamin, fimbrin, villin)
2) Severing proteins (gelsolin, cofilin)
 Modification of filament elongation:
Actin-binding proteins
• Thymosin
• Binds G-actin
• Prevents F-actin
assembly

• Profilin (competes with thymosin)

• Binds G-actin
• Promotes filament assembly
at + end (actin-ADP→ actin-ATP)

• Cofilin
• Promotes disassembly at (-) end

• Gelsolin
• Caps (+) end (prevents further growth)
Actin-in vivo view
25. Actin Cross-linking proteins
• α-actinin
• Loose packing of
bundles
• Stress fibres
• Contractile
• Connected to ECM at focal
adhesions (footholds) via
integrin
• Focal adhesions allow cell to
push actin network forward:
movement
• Fimbrin
• Tight packing of
bundles
• Microvilli
• Non-contractile
Actin-Anchors
• Dystrophin: anchors
actin cytoskeleton to the
sarcolemma (muscle cell
membrane) in skeletal
muscles
• Vinculin & Talin: attach
actin cytoskeleton to
membranes in focal
adhesions
Actin-Networks
• Filamin
• makes 3D crosslinks→
web of filaments under
the plasma membrane
(support)
• Spectrin
• Forms 2D web (held
together by short actin
filaments) under plasma
membrane
• Spectrin network linked to
the membrane by ankyrin
which binds band 3 protein
(integral protein)
• This weblike structural
support allows cells to
deform in capillaries &
spring back unharmed
 26. Cell Movement

• Rho GTPases (rho, rac, cdc42) located at cell surface activate nucleating proteins (arp2/3
complex & formins)
• Lamellipodia (rac)
• Filipodia (cdc42)
• Stress fibres (rho)
• Mediated by actin polymerisation & depolymerisation
• Rho GTPases activate Arp2/3 complex → actin polymerisation at leading edge
• Cofilin also activated by Rho → actin depolymerisation at trailing edge & reorganisation of leading
edge
• Actin-polymerisation & branching (Arp2/3) at leading edge pushes cell forward, stretching actin
cortex
• Contraction (myosin motor protein) at rear propels the body forward to relax tension
• New focal contacts (integrins bound to ECM) are made at front, old ones are disassembled at the
back as the cell crawls forward
• Repeat
• Thus, RAC initiates lamellopodium and RHO mediates the formation of new focal contacts
27. Wiskott-Aldrich syndrome
• Severe X-linked immunodeficiency syndrome

• Mutation WASP (expressed only in haematopoietic cells)

Rac-GTP / cdc42-GTP

WASP

Arp2/3 activation

→ failure of actin nucleation & disruption of signalling pathways

• Death before adulthood without BMT

28. Drugs for Actin- These inhibit cell movement

• Cytochalasins (excreted by fungi):

• blocks depolymerisation

• Phalloidin (toadstool mushroom):

• blocks depolymerisation

• Latrunculin (extracted from sea sponge):

• binds free monomers
29. Myosin
• Contractile activity in
muscle & non-muscle
cells
• 2 ATPase heads (energy
from hydrolysis drives
contraction)
• Tail-tail interactions →
large bipolar thick
filaments
30. Contractile Unit
• Z disc = cap Z & α-actinin
• ATP hydrolysis drives the power
stroke.
• Rigor Mortis results from no ATP and
Calcium ion being abundant in the
cytoplasm rather than the
sarcoplasmic reticulum.
31. Dilated cardiomyopathy
• 1 in 2500, (30% hereditary)
• 1 Mutation = actin (cardiac muscle isoform)
• Mutated where binds to Z disk
→ defective transmission of force in
cardiac myocytes → early heart failure

• symptoms of CHF develop gradually

• dyspnoea, weakness, fatigue,
palpitations, ankle oedema….
• risk of PE & sudden death
32. Familial hypertrophic cardiomyopathy
• Autosomal dominant
• 1 in 500
• Mutations: cardiac isoforms –
myosin II (70%), tropomyosin,
troponin

• #1 cause sudden cardiac death

in athletes

• Asymptomatic/ mildly symptomatic

• Dyspnoea
• Angina
• Palpitation
• Syncope
• Fatigue
• Fatal cardiac arrest (5-10%)
33. Muscle Dystrophies-Theory
• Dystrophin links actin-cytoskeleton to integral
membrane glycoprotein complex that interacts
with ECM (laminin, collagen) Transduces
contractile forces from intracellular sarcomeres
to ECM
• Binds actin, dystroglycans, synaptrohins, dystrobrevin
• Attaches actin-cytoskeleton to plasma membrane & ECM
• Links actin cytoskeleton to laminin-2
• ↑ serum creatine kinase
• Damaged/diseased muscle is repaired by satellite cells & side
population cells
• Satellite cells lie quiescent in basal lamina of mature muscle
(myotubes)
• Stress/trauma : activated → myoblasts
• Attach to surface of myotubes
• Fuse with myofibers to form mature muscle
• Most muscle regeneration
• Stem cells (side population cells, pericyte-derived cells)
• In bone marrow / skeletal muscle vessel walls
• Differentiate into haematopoeitic & satellite cells
• Satellite cells: shortened telomeres: limits reproductive capacity
Dystrophin-Assocated Glycoprotein Complex
• Dystroglycan complex

• Sarcoglycan complex

• Cytosolic adapter protein

complex:
• Dystrophin
• alpha-actinin
• Syntrophins
• Grb-2 Absence of dystrophin
• NOS → loss of DAP at sarcolemma
• Caveolin-3 Absence of physical link
→ fragile sarcolemma
•Muscle degeneration during repeated cycles
of contraction & relaxation
 X-linked
Muscular Dystrophies
 Duchenne MD (DMD)
• Most common & most severe degenerative skeletal & cardiac
muscle disorder

• Incidence: 1 in 3500 males worldwide

• Genetically lethal in males

• 2/3 mutation from carrier mother
• 1/3 new mutation; 10-20% gonadal mosaicism

• Xp21, 427KDa dystrophin mutation:

• Partial deletions (≥ 1 exons) (65% cases)
• Frameshift insertion/deletions (96%)
• Point mutations
→ truncated, non-functional protein
• No detectable dystrophin in muscle Dystrophin Staining
 Dystrophin

• DMD: total loss of function

• BMD: partial function

• Largest known gene (spans 2.3 Mb, 14kb encoding)

• 7 promoters (tissue-specific transcripts), alternative splicing
• 79 exons
• Interspersed intronic repetitive sequences (high incidence of
mutation)

• Binds actin cytoskeleton to sarcoglycan-dystroglycan complex in

muscle cells
 Dystrophin

• Slightly shorter
protein can have
some function
(BMD)
 DMD: Clinical presentation
• Normal at birth

• Develop muscle weakness

by age 3 - 5 (begins pelvic
girdle muscles, progresses
to shoulder girdle muscles)

• Leg weakness: Gower’s sign

• Waddle-like gait

• Calf pseudohypertrophy
(repeated muscle damage)

• Lordosis, scoliosis
DMD
 DMD: Clinical presentation, cont.

• Muscle atrophy → wheelchair bound by

10-12yrs

• Muscle contractures

• Mild cognitive impairment - (20% ↓IQ)

• Respiratory & cardiac musculature

increasingly impaired

• Rarely survive beyond 20-30yrs:

respiratory failure (70%) or
cardiac failure
 DMD / BMD diagnosis
• ↑ serum creatine kinase (CK) levels

• Muscle biopsy :
• Immunohistochemistry & western blot
(anti-dystrophin Abs)

Normal muscle dystrophin isoform = 427 kDa

a + c: control
b + d: DMD
(b: abnormal variation in fibre size,
degenerating & regenerating fibres,
fibrosis
d: no sarcolemmal staining)
 PCR Deletion Screen

• For Confirmation of DMD Diagnosis

• 18 exons: 98% deletions
• Below: 9 exons selected from dystrophin gene of 20 affected boys
• Gel electrophoresis of PCR amplified DNA
 DMD carriers

• Most: no clinical manifestation of disease

• 8% female heterozygotes (carriers) have muscle weakness

• Manifesting heterozyotes
• random X-chromosome inactivation
 Becker MD (BMD)
• 6 in 100,000
• Mutation dystrophin: inframe insertions / deletions
→ aberrant but partially functional dystrophin protein
→ more mild disease form

• 15% of dystrophin mutations

• Onset late childhood / adolescence

• Slower progression & significant variability
• Mutations used to identify “important”
dystrophin domains
 Potential Treatment
Loss of exon

X
• Since:
• DMD= Deletions, Frameshift, and Nonsense mutations
causing an absence of Dystrophin
• BMD= in-frame deletion resulting in a partially
functional dystrophin.
Potential treatment
- Trick splicing machinery into skipping exons with
nonsense mutations or translation machinery to read
through PTCs ameliorate DMD to BMD
 Emery-Dreifuss MD (EDMD)
• 1 in 100,000
• X-linked (also autosomal dominant & recessive forms, but rare)
• Mutation Emerin (or LaminA/C)

• Defects in lamin (IF) assembly / attachment to nuclear envelope

→ fragile nuclear envelope
affects physically stressed tissues: muscle fibers
 EDMD: Clinical presentation

• Onset: early childhood

• Affects skeletal & cardiac muscle

• Early contractures: elbows, ankles, neck

→ flexion deformity of elbows
→ reduced joint mobility

• Slowly progressive muscle weakness &

atrophy (upper arms, lower legs, progress
proximally to shoulder & hips)

• Cardiomyopathy, Conduction defects &

arrhythmias in adulthood - pacemaker by 30yr
• Sudden cardiac death common
 Autosomal
Muscular Dystrophies
 Myotonic Dystrophy
• Autosomal dominant
• Common (1 in 8000, 2nd after DMD)
• myotonin protein kinase (MD type 1: 98%)
• Trinucleotide repeat disorder (CTG): anticipation
• Normally <30 repeats. Severely affected several thousand

• ? Pathogenesis

• Multisystemic disorder
 Myotonic Dystrophy: Clinical presentation
• Onset 20-40 (any age: birth to old age)
• Slowly progressive muscle degeneration & myotonia
• Myotonia = sustained involuntary contraction of muscle group
• Weakness: hands, legs (gait abnormalities), sternomastoids
• Atrophy of facial muscles
→ ptosis & haggard appearance

• Other:
• Cataracts
• Frontal balding
• Gonadal atrophy
• Cardiomyopathy
• Endocrine abnormalities (DM)
• Cognitive & behavioural problems
• Dementia
 Facioscapulohumeral MD

• Rare

• Autosomal dominant

• Progressive weakness of
face, scapula, upper arms

• Deletion of subtelomeric
tandem repeat (4q35) or t4q;10q

• ? pathophysiology
 Facioscapulohumeral MD: Clinical presentation
• Age onset: 10-40 yrs (95% by age 20)
• Inability to puff cheeks
• Initially facial weakness (expressionless), ptosis
• Weakness: shoulders, upper arms
• difficulty raising arms → scapular winging

• Eventually progresses to legs

• Sensorineural hearing loss
• Arrhythmias

• Normal life expectancy

 Limb Girdle MD (LGMD)

• LGMD 1 (autosomal dominant)

• Rare, Adult onset
• Laminopathies (allelic with
autosomal dominant EDMD)
• Caveolin-3 (PM invaginations:
regulate signal transduction
→ Sarcolemmal membrane alterations

• LGMD 2 (autosomal recessive)

• Onset: 10-20yrs
• Calpain
• α-, β-, γ-, δ- sarcoglycans
• Titin (associated with myosin)
• Telethonin
 Sarcoglycanopathies: LGMD

Sarcoglycan Complex:
• N-glycosylated transmembrane
proteins (glycocalyx)

• Linked to dystrophin via

association with dystroglycan
complex

• Defective assembly of sarcoglycans

→ disrupted interaction with dystroglycan protein complex
→ disrupted association of sarcolemma with ECM
 LGMDs: Clinical presentation

• Clinically similar to X-linked DMD

• Weakness of proximal musculature
• “Limb girdle”: hip & shoulder

• Onset age 10-20

• Slow (& variable) rate of progression
• earlier onset, faster progression

• Severe disability within 20-25 yrs

• Death: respiratory failure
α-sarcoglycan
• LGMD vs. DMD: staining
• Pseudohypertrophy & contractures rare
• No cognitive impairment
 Congenital muscular dystrophy
• Onset: birth

• Mutation: Laminin (skeletal

muscle isoform) & ……

• General muscle weakness,

respiratory insufficiency,
contractures, seizures,
mental retardation

• impaired myogenesis, synaptogenesis &

mechanical stability

• Clinically variable Pectoral folds (Arrow) with

severe shoulder weakness
Muscular dystrophies: Summary
 34. Intermediate filaments
• Important for tensile
strength.
• It forms the nuclear
lamina
• Desmosomes and
hemidesmosomes.
• The origin of cancer cells
could be detected from
IFs in the case of
metastasis.
Intermediate Filaments-types
35. IF Diseases
36.Plasma MembraneComponents
• It is Asymetric
• Fungal PMs contain Ergosterol
• Cholestrol increases membrane stability more rigid
• Asymmetry: maintained by flippases
• Functional importance
• Phosphatidyl serine flipped to outer leaflet during apoptosis
• Flip-flop requires
• flippases
• phospholipid specific
• scrambalases
• non-specific scrambling
• in ER membrane: mix up newly synthesised PLs
• at plasma membrane, activated during apoptosis
 Red Blood Cell Membrane

• No nucleus / organelles
• 4-5 tetramers of spectrin (α & β chains) linked into a junctional
complex held together by binding short F-actin & protein 4.1
• spectrin network reinforces bilayer; deformable network
→ can withstand stresses
• Ankyrin binds band 3; attaching spectrin cytoskeleton to membrane
• Protein 4.1 links actin to glycophorin (single pass transmembrane
glycoprotein), also binds spectrin & band 3
• Band 3 : multipass transmembrane protein
Plasma Membrane-Acanthocytosis
• Occurs in alcoholic patients with Portal
HTN.
• It is a spur cell anemia associated with
chronic liver disease.
• XS cholesterol transferred to outer leaflet
→ formation of flat, scalloped cells with
projections → ↑ surface area of outer
bilayer → Less deformable

• ↓ deformability → sequestration
& destruction by spleen
→ haemolytic anaemia
 Hereditary Spherocytosis
• Autosomal dominant (75%)
• RBC cytoskeletal membrane defect
• non-functional skeletal membrane protein;
spectrin, ankyrin or protein 4.1
→spectrin deficiency
unstable membrane, loses fragments
→RBCs spheroidal, ↓ deformability
→ Vulnerable to splenic sequestration Spherocytes: small & no area
& destruction of central pallor

→ Haemolytic anaemia
• Splenomegaly
• Jaundice
• Gall stones (hyperbilirubinaemia)
• Increase in MCHC, RDW

• Tx: folate supplement, splenectomy

Plasma Membrane-Lipid Rafts and Membrane Proteins

• Lipid rafts are cholesterol and

glycosphingolipids (long saturated tails) rich.
Thus, they are more sable and less fluid.
• Normally stick out of lipid bilayer due to the
longer and straight tails involved in their
formation.
• Contain a high concentration of clustered
integral and peripheral proteins. They also
contain GPI (Glycosylphosphotidylinositol)
anchored proteins only on the ECM side.
• Lipid rafts cluster proteins together so they
could carry out functions together or for
receptor mediated endocytosis.
• GM1 Gangliosides are receptors for the
cholera toxin found in intestinal epithelial
cells.
• Proteins:
• Integral: amphipathic
• Peripheral: noncovalently attached [Cyt C]
• Lipid-anchored (peripheral): either sides of
plasma membrane see figure on the side)
 Cell Walls vs. Cell Membranes
Prokaryotes have both walls and membranes. Eukaryotes, at least animals,
Walls = Carbohydrate (peptidoglycan) only membranes.

Pink

Cell A
Cell B

Purple
= glycocalyx

Carbohydrates on surface, but not

organised into a wall
37. Sodium-Potassium Pump
• 3 Na+ out and 2 K+ in for
each ATP hydrolyzed on the
cytoplasmic side.
• Oubain inhibits the channel
via binding to the K+ side.
• Cardiac Glycosides (digoxin
and digitoxin) directly
inhibits the Na+/K+ ATPase
• This results in an indirect
inhibition of the Na+/Ca2+
exchanger where the would
be an increase in cytoplasmic
Ca2+ and this leads to an
increase in cardiac
contractility.
38. Collagen-Types
• This is the MOST abundant protein in the human body.
Modified via posttranslational modification. It organizes to
strengthen the ECM.
• Matrix Metalloproteinases destroy collagen
• Type I: Bones [made by osteoblasts]; skin; Tendon; A
decrease in production causes osteogenesis Imperfecta type
1; cornea; late wound repair.
• Type II: Cartilage/Hyaline, Viterous body, nucleus pulposes
• Type III: Reticulin-notably found in blood vessels
• Vascular type of Ehler-Danlos Syndrome
• Type IV: Basement Membrane, lens, basal lamina.
• Defective in Alport Syndrome
• Autoantibodies against Type IV collagen in Goodpasture syndrome
 Collagen-composition
• Made by fibroblasts (osteoblasts – bone,
chondroblasts – cartilage) & epithelial cells (BL)

• Each α-chain is twisted into helix of 3 residues/turn;

stabilised by interchain H bonds

• Glycine every 3rd amino acid

= Small, allows for H-bonding between glycine
backbone with adjacent helix

• Proline & hydroxyproline ~25%; makes kinks, fixed

angle; helps helix formation.

• Also contains Lysine & hydroxylysines

• Triple helix - extremely strong

– Like cables of suspension bridge
– Resists unwinding
39. Collagen-Synthesis

Lysyl Oxidase (Cu+)

40. Collagen defects : Disorders

•Collagen mutations → connective tissue defects

Osteogenesis Imperfecta Collagen I

Ehlers-Danlos syndrome Collagen I, III, V
Alport syndrome Collagen IV
Goodpasture syndrome Collagen IV
Epidermolysis bullosa Collagen VII
 Osteogenesis Imperfecta (OI)
[brittle bone disease]
• Bone: = Type 1 collagen embedded in
calcium phosphate matrix

• Mutations in Type I collagen;

procollagen a (I) chain genes

• Most clinically severe phenotypes from

substitutions of invariant glycine
OI Type II
• Triple helix forms C N-terminus
gly mutations near C-term’ = more
deleterious; N-term’ permit substantial
triple helix formation

• Range of phenotypic severity:

• Perinatal lethal
• mild predisposition to fractures
• Often mistaken for child abuse because
of recurrent fractures
OI Type III
Osteogenesis Imperfecta OI Type I

Dentinigenesis imperfecta due to lack of Dentin

• Skeletal deformities, Fractures (bone fragility), blue sclera due to
translucency of the connective tissue over the choroidal veins.
OI Type Clinical Manifestations
I (mild) blue sclerae, bone fragility

II (perinatal lethal) blue sclerae, severe bone fragility, no mineralisation,

skeletal deformity

III (deforming) blue-white sclerae, dentinogenesis imperfecta,

bone fragility, short stature

IV (mild deforming) normal sclerae, dentinogenesis imperfecta,

mild short stature, moderate skeletal fragility
 Osteogenesis Imperfecta
• This is an AD disease with a
decrease in Type I collagen.
• Hearing results due to
abnormal ossicles
• Type I - null mutations,
haploinsufficiency
• Types II-IV - missense,
dominant negative
• Some cases are AR because of
collagen modification defect
- severity approximately
correlated with size of mutant
amino acid and position of
substitution
• Bisphosphonates - as used for
osteoporosis - potential
treatment for OI-type I
 Ehlers-Danlos Syndrome
Can be AD or AR disease
• Abnormal fibrillar collagen structure
• Connective tissue weakness: Lacks adequate tensile strength
→ hyperextensible skin (fragile, susceptible to trauma/bleeding)
→ hypermobile joints (dislocations)

• May also have internal complications

• rupture of colon
• rupture of large arteries

• Other symptoms
• varicose veins, ecchymoses
• Berry’s or Aortic Aneurysims
 Ehlers-Danlos Syndrome
Clinically & genetically heterogeneous group of disorders:
defective fibrillar collagen synthesis or structure
(>10 variants known)

• Type I, 43% EDS (severe), Type II, 35% EDS (mild): Collagen I / V mutation
• Joint hypermobility, hyperextensible skin [most common type]

• Type IV, 6% EDS: Collagen III mutation

• Collagen III found in blood vessels & granulation tissue
• Vascular type: arterial, intestinal, uterine rupture, easy bruising, thin
translucent skin, varicose veins. + organ rupture

• Type VI, 2% EDS: Mutations in lysyl hydroxylase

• Collagen lacks structural stability
• Ocular fragility: retinal hemorrhage and detachment, corneal rupture
• Kyphoscoliosis

• Type VII, 3% EDS: Defect in converting procollagen I to collagen I

• Joint hypermobility
 Alport Syndrome
• Common inherited cause of kidney failure

• Mutation α5 chain of Type IV collagen (basal lamina)

• Nephritis & deafness

• Haematuria, Proteinuria, HTN
 Goodpasture Syndrome
• Rare auto-immune disease (onset: teens-20’s & ↑ males)
• Auto-antibodies to Type IV collagen (α3 chain)

→ Inflammatory destruction of BM in kidney glomerulus & lung alveoli

• Haemoptysis & glomerulonephritis with progressive renal failure

 Epidermolysis Bullosa
• 3 forms (depth of blister formation in relation to basal lamina)

• EBS (EB simplex): mutation keratin 5 or 14

• Junctional EB: mutation laminin, integrins,
hemidesmosomal protein
• Dystrophic EB: mutation collagen VII

• “butterfly children”: extremely fragile skin,

minor mechanical friction / trauma recurrent blister formation
 Dystrophic Epidermolysis Bullosa

• Mutation collagen VII: absence of anchoring fibrils

• Severly debilitating blistering causes

syndactyly

“the boy whose skin fell off”

(Jonny Kennedy)
 FGFR3 Mutation: Achondroplasia

•Most common form of dwarfism

•Incidence :1 in 10,000 live births

•Mature height <4 feet

•Constitutive expression of FGFR3 in

chondrocytes
→ restricts chondrocyte proliferation
(bone growth)

→ excessive bone growth inhibition

 FGFR & Tyrosine kinase signalling

•FGFR negatively regulates bone growth

inhibits chondrocyte proliferation inhibits cartilage growth; type II collagen

•Constitutive activation of the RTK, FGFR3 dwarfism

Menkes Disease
• This is a connective tissue disease caused by an
impared copper absorption and transport. This
will lead to a decrease in lysyl oxidase (requires
Cu+ as a cofactor).
• Brittle, “kinky” hair , retardation, and hypotonia
• steel fuzzy sparse hair
• X-linked recessive inheritance
• ATP7A gene is mutated
• provides instructions for making a protein that is
important for regulating copper levels in the body
41. Elastin-Overview
• Stretchy protein found in the arteries, skin, lungs, vocal
cords, elastic ligaments, uterus, and ligament flava of the
vertebra.
• Rich in proline and glycine the nonhydroxylated form.
• Tropoelastin with fibrillin scaffolding
• Fibrillin binds to elastin & is essential for assembly & integrity of
elastic fibers
• Crosslinking gives elastin the elastic property it has.
• Elastase breaks Elastin down that is inhibited by α1-
antitrypsin ( this also inhibits trypsin) a deficiency +
smoking that recruits Neutrophil that produce elastase
results in COPD
• Wrinkles the result of aging are due to a decrease in
collagen and elastin production
 Marfan syndrome:
Fibrillin mutation (AD)
Common connective tissue disorder
(1 in 5000 worldwide) Weak elastic tissue;
•Fibrillin is a glycoprotein forming a sheath around elastin
•Heart: aortic root dilatation & dissection
aortic/mitral regurgitation
Cystic medial necrosis of aorta aortic incompetence and
dissecting of the aortic aneurism
floppy mitral valve
•Eye: lens subluxation [upward and temporally] & retinal detachment
•Skeleton: tall & thin
long limbs & fingers (arachnodactyly)
funnel chest (pectus excavatum)
Hypermobile joints
Arachnodactylyl
- FBN1 mutation in chromosome 15 defective fibrin=elastin’s scaffold
connective tissue disorder.
 Emphysema
• COPD

• Dyspnoea, hyperventilation
• Hyper-inflated chest (barrel chest)
 42. Nucleus-Export and Import via Nuclear Pore Complex (NPC)
IMPORT
• Cargo protein with NLS binds to a/b importin
• Importin-cargo complex interacts with nucleoporins
(glycoproteins of Nuclear Pore Complex[NPC]), translocates
complex inside
• Nuclear Ran-GTP interacts with importin b, dissociating the cargo
complex
• RanGTP -bound importin transported back to cytoplasm
• Ran GAP :
RanGTP→ RanGDP,
releases importin
EXPORT
• Ran-GTP bound exportin → binds to cargo & nucleoporins,
activates translocation through NPC
• Cytosolic Ran-GAP :
RanGTP → RanGDP
→ exported cargo detaches
• Ran-GDP and exportin
move back into nucleus
• Nuclear Ran-GEF catalyses
GDP/GTP exchange
NPC:
- It contains Neucleoporins
- 8 subunits
 Nuclear Lamina and Mitosis
• It is a network of proteins where lamin
is intermediate filaments.
• Lamins A, B, C form meshwork
• They bind to emerin and other proteins
• Lamin B: LMNB gene
• Lamin A & C: LMNA spilce varients
• Lamin phosphorylation during
prophase by Cdk1
→ nuclear lamina
disassembly
→nuclear envelope
disassembly into vesicles containing
lamin B

• Lamins A & C released as free

dimers
• Lamin B = anchored to inner
membrane

• Inactivation of Cdk1→
lamin dephosphorylation
• Membrane vesicles bind
chromosome surface
→ reassembly
 Subnuclear and nucleolus substructures
• Fibrillar center
• Transcriptionally inactive DNA
• NORs (nucleolar organiser regions
• =where pre-rRNA genes located
• Dense fibrillar components / pars fibrosa
• rRNA being transcribed then cleaved & modified
by snoRNPs
• Granular component / pars granulosa
• rRNAs begin assembly with ribosomal proteins
• NOR: contain 5.8S,18S, and 28S as tandem
repeats
• Clustered on 5 chromosomes (13, 14, 15, 21, 22)
• Transcribed by RNAP I

Cajal Bodies
 Spinal Muscular Atrophy

• 1 in 40 carriers
• SMA types I-III (infantile, intermediate, adult)

Gems contain SMN (survival of motor neurons) protein

• Mutation SMN → defective snRNP assembly & defective
pre-mRNA splicing → loss motor neurons (spinal cord &
brainstem)

Generally: sudden onset, rapid progression

• Muscle weakness & atrophy
• Hypotonia
• Dysphagia & feeding difficulties
• RTIs
43. LAMINOPATHIES
 Skeletal & Cardiac Myopathies
• Emery-Dreifuss muscular dystrophy
• Mutation Emerin or LaminA/C
• Contractures, especially in the elbows,
ankles, neck → Flexion deformity of
elbows, limited neck flexion
• Muscle weakness & atrophy
• Conduction defects & arrhythmias
• Sudden heart failure common

EM from skeletal
muscle from an
EDMD patient
Arrow: NE is
disrupted
Arrowhead:
chromatin is
extruded into the
cytoplasm
 Skeletal & Cardiac Myopathies

• Dilated Cardiomyopathy
• Lamin A/C defect (rare cause)
• Fragile nuclear lamina → nuclear structures/contents damaged
→ cell death
→ congestive heart failure
 Lipodystrophy

• Lamin A/C defect

• preLamin A interacts with adipocyte TF

→ impaired adipocyte differentiation

• Accumulation of adipose tissue in face and

neck

• Peripheral lipoatrophy with muscle prominence

 Hutchinson-Gilford Progeria Syndrome
• Accelerated aging in children
• ~ 1 in 4 million
• Autosomal dominant - sporadic
• Altered lamin A →unstable NE (bleb formation, loss peripheral
heterochromatin, NPC clustering)
→ progressive nuclear damage
→ cells die prematurely
44. Mitochondria
• Aerobic respiration and energy production (ATP)
• Requires an electrochemical gradient
• Involved in Apoptosis
• It is located in energy requiring locations of the cell.
• Outer membrane is permeable
• It synthesizes cardiolipid
• It is 20% of inner membrane and impermeable to
ions
• It contains porins
• Inner membrane is impermeable
• ETC complex and import receptors for matrix proteins
• Thermogenin (UCP) allows protons to “leak” from
cytoplasm to matrix does not produce TAP and it is
found in brown adipose tissue to produce heat in a non-
shivering manner
• uncouples gradient: not need to use ATP synthase
(Uncouples respiration from ATP synthesis)
• Oxidative metabolism (TCA, ETC) produces HEAT
shivering
• Reactive Oxygen Species (ROS) are bad and damaging
but SOD2 and GPX get rid of them
• Histologically:
- Lamellar Cristae: Most Cells
- Tubular Cristae: Steroid Secreting
 Barth syndrome
• X-linked cardiolipin synthesis disorder

• Cardiomyopathy
• Generalised muscle weakness & chronic fatigue
• Neutropenia
Healthy mitochondrion Barth mitochondrion

• High mortality in infancy:

• Sudden infant death
• Infection
• Cardiac failure
 Mitochondrial pore formation & Apoptosis

Pro-apoptotic bcl-2 proteins (BAD,

BAX) translocate from cytosol to
mitochondrial membrane in response
to cell damage/stress & signalling
↓
Formation of pores in outer
membrane (BAX)
↓
Release pro-apoptotic factors
(cytochrome C) into cytosol
↓
Activation caspase cascade
↓
Apoptosis
 Protein import: TOM & TIM
• Hsp70+protein binds to TOM
(translocase of outer membrane)
TOM recognises mt signal sequence

• Precursor translocated across inner

membrane via TIM
(translocase of inner membrane);
requires electrochemical gradient

• Signal sequence removed by matrix

protease

• Protein spontaneously folds or

helped by chaperone Hsp60 & ATP
 45. Mitochondrial Genetics
The genome:
• Circular double stranded structure of 16569 base
pairs
• The DNA strands are called H and L based on their
sedimentation velocities in the ultracentrifuge (H: G-
rich, L: C-rich)
• 2 rRNA, 22 tRNA, and 13 proteins genes [Complex I,
IV, V, and part of III]
• The H-strand is all coding (except for the D-loop)
• The L-strand codes for 1 mRNA and several tRNA’s
• Only 13 (!) for protein coding genes.
• tRNA genes are dispersed flanking most other genes
• The only noncoding region is called displacement
loop, aka control region
• Origin of replication for the H-strand
• Promotors for transcription of both the H-strand and the
L-strand
• Mitochondrial txn
• No capping
• Yes poly(A)
• Heteroplasmy: more than one type of organelle
genome
DNA Polymerase γ [POLG] & [TWINKLE]= mtHelicase

• Myopathies due to AZT treatment of HIV

• DNA Pol γ is the only one inside the
mitochondria; thus, it has a repair function
too. It is affected by Zidovine (AZT). AZT
does not affect cellular DNA pol δ.
Progressive External Ophtalmoplegia (PEO)
• mtDNA depletion (reducing its levels) or
multiple large deletions in the mtDNA
• Autosomal dominant PEO is mostly caused
by mutations in the polymerase domain of
DNA Polymerase γ, but can also be caused
by mutations in TWINKLE, the gene for the
mitochondrial helicase.
Medical Genetics-Mitochondria
• PEO
• Kearn-Sayer Syndrome (KSS)
• This is a disease of late onset and the mtDNA deletion found in muscles. Without bone marrow involved.
• Pearson’s Syndrome
• With bone marrow involvement. Occurs in pediatrics. Affects all tissues. Some patients switch to KSS
phenotype.
• MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes)
• Mutated gene: tRNALeu (A3243G; T3271C; A3251G)
• always heteroplasmic
• MERRF (Myoclonus Epilepsy and Ragged Red Fibers)
• Mutated gene: tRNALys (A8344G; T8356C)
• always heteroplasmic (so homoplasmy for the mutation is probably lethal)
• LHON (Leber's Hereditary Optic Neuropathy )
• Mutated genes: Missense mutations in subunits of Complex I, (G11778A, Arg340His in subunit 4;
T14484C, Met64Val in subunit 6; G3460A, Ala52Thr in subunit 1)
• may be homoplasmic (but still the optic nerve is the only affected tissue !)
• NARP(Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa)
• Mutated gene: ATPase 6 gene (T8993G/C, Leu155Arg in subunit 6 of Complex V)
• always heteroplasmic (so homoplasmy for the mutation is probably lethal)
• maternally inherited Leigh Syndrome is a more severe form with a larger proportion of mutated mtDNA
All of these mutations in Mitochondria require a threshold until they are actually expressed as a
disease in an individual
46. Cellular Signals-Overview
 • Using cAMP
Cellular Signals- GPCR • Using DAG and IP3
Effects of bacterial exotoxins on signal transduction
•Cholera toxin (Vibrio cholerae):
ADP ribosylation (toxins attach ADP-ribose residue
to Gα subunit of Gs) Locks stimulatory Gs into the active state by
blocking GTPase activity
→ Activates secretion pathways
•E.coli toxin, Traveller’s disrrhoea: mechanism same as Cholera.
•Pertussis toxin, whooping cough (Bordetella pertussis):
ADP ribosylation Locks Gi into GDP-bound state;
inactivates Gi
→ Prolonged & inappropriate response
All cause ↑cytosolic cAMP & signaling, but different mechanism

Gq
 Defects in GPCR signalling
Normal retina

•Retinitis pigmentosa 4 :

Mutation Rhodopsin (rod pigment)

degeneration of photoreceptor cells

in retina

Loss / diminished night & peripheral vision

Tx: Vitamin A slows progression

 Activated Receptor Tyrosine Kinases
• Activated RTKs Phosphorylate effector proteins;
1. PLC
Breaks down membrane inositol phosphates to 2nd messengers;
1. IP3 Ca2+ (released from ER) activation PKC
2. DAG activation PKC activation TFs
2. PI3K (Phosphatidyl-inositol 3-kinase)
Phosphorylates membrane phospholipid
Products Activation PKB cell proliferation & growth
inhibition apoptosis (survival)

• Activation reveals docking site for proteins (GRB2)

3. GRB2 binds SOS (GEF) activate small G-protein (Ras)
activates mitogen activated protein kinase (MAPK) pathway
TFs (Fos & Jun)
production GFs, GFRs, cell cycle proteins
cell proliferation & growth
AKT/PKB promotes cell survival
PKB inhibits BAD
BAD inhibits BCL-2
BCL-2 inhibits BAX
BAX forms pores

PKB PKB
 Sos and GRB2 system in Insulin

•Receptor monomers linked by disulfide bonds (insulin

receptor)
•Autophosphorylate their tyrosine residues
•Use Grb2, Sos, & Ras mediators & stimulate transcription
•Insulin signalling also works in cytosol to alter metabolic
activity & cause fusion of vesicles filled with glucose
transporter (GLUT) to plasma membrane
→ glucose uptake
 Cancer & NIDDM due to RTK defect
NIDDM; Non Insulin-Dependant Diabetes
Mellitus
• Downregulation of insulin receptor
• ↓ receptor phosphorylation & TK activity
• ↓ 2nd messengers in pathway
• → Insulin resistance in skeletal
muscle, liver, adipose tissue
• Pancreatic β-cell dysfunction → insulin
deficiency
JAK/STAT system (Cytokine Receptors) + Integrin Receptors
Receptors without kinase activity
• Cytokine receptors
• Like RTKs, but no TK
• Ligands= ILs, G-CSF, GF
• Ligand binding activates kinase
domains on proteins (JAK) that are
associated with them
• JAK/STAT pathway (Signal
Transducers & Activators of
Transcription)
Integrin receptors
• Ligand binding→clustering of
receptors which recruits & activates
kinases
• ILK (Integrin-linked kinase)
• FAK (Focal adhesion kinase)
• Cooperative Integrin & RTK
signalling controls migration
• proliferation, growth, differentiation,
survival, apoptosis
 Steroid Hormone Receptors
• Hormones bind to
Intracellular nuclear
receptors
• Receptors bind to HRE
(Hormone
• Responsive Element) in
DNA sequence
• Receptors = transcription
factors
• Receptors also bind Zn2+
= essential for DNA
binding
47.Cell-Matrix Interactions
• Laminin and fibronectin (adhesive
glycoproteins) bind to different
integrins (transmembrane cell
receptors)
• Via RGD
• Focal adhesions: Integrins bind to
actin stress fibers via adapter
proteins, and to fibronectin
• Hemidesmosomes: Integrins bind to
intermediate filaments (keratins) via
adapter proteins, and to laminin
• Bullous Pemphgoid: an autoimmune
blistering disorder due to antibodies
against hemidesmosomes proteins
• Pemphigus vulgaris: an autoimmune
blistering disorder due to antibodies
against desmosomes proteins
 Extracellular matrix Organization
• Fibers - embedded in proteoglycan gel
• Collagen (strengthen, resists stretching & organises ECM)
• Elastin (elasticity)
• Fibrillin
• FACIT: Fibril-Associated Collagens (VI, IX, XII)
with Interrupted Triple helices
• Organise fibrils in the ECM: Mediate interactions of collagen fibrils with
other fibrils & ECM proteins, proteoglycans & GAGs
• Bind surface of fibrillar collagens
• 3-stranded structure interrupted by nonhelical domains
→ more flexible than fibrillar collagens
• Ends not cleaved after secretion, retains propeptides
• Ground Substance - hydrated gel, resists compressive forces,
permits rapid diffusion of nutrients, metabolites & hormones
between blood & tissue
• Proteoglycans (protein + GAGs): Resist compression & fill
spaces
• Hyaluronan is a unique Glycosaminoglycan. It does not bind to a
protein + non-sulfated
• Could be found in vitrous humor
• Glycoproteins (sugars + protein) : Adhesive glycoproteins
• fibronectin, laminin, entactin
 Basal Lamina and Diabetic Nephropathy
network of;
• Type IV Collagen
• Laminin
• Entactin / Nidogen
• HSPGs – Perlecan
• Collagen IV binds to laminin forming scaffold. Entactin &
Perlecan bind to both Laminin & Collagen IV, connecting the
2 networks.
• Perlecan (heparan sulfate proteoglycan; HSPG) is found in all
basement membranes
• Perlecan mediates cell attachment to basal lamina for
endothelial cells and fibroblasts
• Perlecan binds entactin, laminin, collagen IV &
fibronectin
• Interactions anchor proteoglycan in matrix, increases
its strength
• HSPGs perlecan & syndecan function in angiogenesis &
mitogenesis
• Diabetic Nephropathy: due to a basement membrane
dysfunction because GAGs such as HSPG perlecan function
as a size and charge selective barrier @ bowman’s
space/capsule
 Fibronectin: Soluble & Insoluble fibrillar forms

• Soluble plasma fibronectin =

plasma glycoprotein, made by liver,
circulates in blood, enhances blood
clotting (& wound healing)

• Insoluble fibronectin fibrils form on

cell surface & deposited in ECM

• Necessary for embryogenesis-

guides cell migration
Neural crest cells migrating out
from chick nervous system
guided by fibronectin-coated
surface
48. Cell-Cell Interactions Transmembrane adhesion proteins : Claudin &
Occludin.
•extracellular domains join directly
•Zonula Occludens (ZOs) are anchored to
actin cytoskeleton
•Clinically: have a role in Chron’s disease
and Ulcerative Colitis both considered
inflamtory bowel diseases that increase
the risk of GI cancer.

Cadherins : require calcium to bind their subunits

together

6 connexin subunits / connexon

2 connexons = 1 channel
dopamine permeability in response to light
 Clinical Cases
• Charcot-Marie-Tooth (CMT) Disease:
• In the form missing connexin-32; these
are necessary for intra-cellular gap
junctions between coils of Schwann cell
that wrap around the neuron. These
intracellular junctions may be critical in
supplying nutrients to the distant
reaches of the Schwann cell; if impaired,
demyelination & failure of peripheral
neuron function.
• This disease is NOT life threatening but
there is no good treatment for it.
• Leukocyte Adhesion Deficiency:
• The patient will suffer from repeated life
threatening bacterial infection due to
impaired chemotaxis and extravagation
of Neutrophils.
• It is due to a β2 integrin mutation. • Selectins on activated endothelial
cells bind oligosaccharides on WBC
• Glanzmann’s Thrombasthenia:
• Low affinity, WEAK ADHESION
• This is a platelet dysfunction due to the • Leucocyte “rolling” → activates it’s integrins
absence of platelet integrin complex
• It is also due to a β3 integrin mutation.
• Integrin binds ICAM (Ca2+ independent CAMs) on
• As a result platelets do not bind to endothelial cells:
fibrinogen and platelets do not
aggregate. • STRONG ADHESION
• Brusing and bleeding results and • Allows WBC migration into tissue: diapedesis
confused with child abuse.
Heridatry Neuropathy with Liability to Pressure Palasies (HNPP) and CMT
• CMT= Genetically heterogeneous because it could
be cause by over 20 loci. Neuropathy causes motor
and sensory polypathy with an autosomal
dominant mutation in CMT1 gene. Hyporeflexes,
and slowing in nerve conduction velocity (NCV).
• HNPP=
Autosomal dominant
Repeated focal pressure neuropathies
(e.g. carpal tunnel syndrome)
Prolongation of distal nerve conduction latencies
Evidence for demyelination on biopsy
--------------------------------------------------------------------------
-Both HNPP and CMT1A map to chr. 17p11.2 (but
distinct from SMS deletion region)
PMP22 - major component of myelin
- expressed in myelinated fibers in PNS
- predominantly produced by Schwann cells
- role during Schwann cell growth &
differentiation
49. PCR
- It is a molecular biology technique used to amplify a desired
fragment.
- Important in neonatal HIV testing & Herpes
Encephalitis
- It uses a Taq polymerase (heat resistant)
- Agrose gel electrophoresis is used to separate the PCR
products
MLPA= Quantitative PCR
Sickle Cell Disease

• It results in a change
of a restriction
enzyme.
50. Blotting Techniques

• SNoW DRoP
• Southwestern blot:
is used to identify
DNA-binding
proteins (TFs) using
labeled
oligonucleotide
probes
 51. Microarrays
• Many nucleic acid sequences are arranged in grids on glass or silicon.
• DNA or RNA probes hybridize to the glass.
• Scanner is used to detect the relative amount of complimentary
binding.
• Profile gene expression of many genes simultaneously.
• Detect SNPs and CNVs for genotyping, genetic tests, forensics, cancer,
and genetic linkage analysis
 52. ELISA

• Indirect: detects antibody

• Direct: detects antigen
 53. FISH
• Detects
microdeletions
(>100kb)
• FISH probes along
entire length of
chromosome – very
good for visualising
rearrangements in
cancer cells
• Spectral
Karyotyping
Using FISH to define chromosomal breakpoint
t(8;16)(p22;q12) Physical map of breakpoint region
14_09.jpg
on 8p

Results of successive FISH expts

54. Cloning methods-this method
expresses recombinant DNA in
mammalian cells
Cloning methods
Cloning methods

Functional Cloning: identify genes by its product

such as factor VIII.

Positional Cloning: identifying genes by its

location such as mapping a disease in
achondroplasia.
 55. Gene Expression Modification
• Cre-lox system:
manipulate genes
at specific
developmental
points such as to
study a gene whose
deletion causes
embryonic death.
 Gene Expression Modification
• Transgenic strategies such as a random
insertion of genes or targeted
insertion/deletion of genes via
homologous recombination
• Also known as Enriching for gene-
targeting events.
• Positive and negative selection to select
for homologous recombination (gene-
targeting)
• An exon from a targeted gene is
disrupted by the a neo gene containing
its own promotor encoding resistance to
the toxic agent G418 (positive selection)
• Downstream of the neo gene is a tk
(Thymidine Kinase) gene, that when
integrated and expressed in the host
genome provides the target for the toxic
agent gancylovir(negative selection)
• Neo+ tk- colonies frequently carry the
desired mutation
RNAi=siRNA=dsRNA
• siRNAs (short interfering RNAs) are Dicer products of
exogenous long double stranded RNA precursors. These
exogenous precursors can originate from natural introduction
in the cell such as viral replication intermediates or from
artificial introduction in vitro or in vivo (gene therapy)
• miRNA’s (micro RNA;s) are Dicer products of genome-
encoded long double stranded RNA precursors
• RNAi is a mechanism that inhibits gene expression (silencing)
by destroying mRNA or inhibiting translation. The mRNA
destruction pathway is favored when there is a perfect match
between the small interfering RNA (siRNA) and the target
mRNA. If the match is not perfect the translation inhibition
pathway is followed.
• Dicer is the enzyme that processes ~ 75 nucleotides long
genome encoded RNAs with profound hairpin structures into
double stranded RNAs (siRNAs) of ~ 23 nucleotides in length.
Dicer does the same for added double stranded RNA
• One of the two RNA strands is incorporated into the RNA-
induced silencing complex (RISC)
• Argonaute is the catalytic component of the RISC complex and
is an endoRNase (slicer)
• Our genome contains about 400 micro RNA (miRNA) genes,
that are transcribed by RNA Pol II and are capped and
polyadenylated before being processed in the nucleus and
further processed by dicer in the cytoplasm. The mRNA’s
whose translation is inhibited are destabilized and end up in
P-bodies (Processing bodies believed to be the destruction
site for mRNAs)
• The destruction pathway can also be initiated in human cells
by just adding double stranded RNA. This is becoming a very
important tool to change gene expression, also for gene
therapy
56. Preparation of a karyotype.

Phytohaemagglutinin stimulates T cell

division

Colchicine prevents spindle formations

so cells arrest in metaphase

Downloaded from: StudentConsult (on 9 August 2006 03:39 PM)

© 2005 Elsevier
57. DNA testing flowchart
Is mutation known?
YES NO

Single nucleotide substitution? Gene known?

YES NO YES NO

Large deletion Sequencing Suspect multiple

ASO
> 100kb? feasible? genes involved?

YES NO YES NO YES NO

FISH Southern blot Sequence Track CGH Exome

mutation sequence
with linked
marker
 Severe haemophilia A
Haemophilia A & Southern Blot is often the result of an
intrachromosomal
rearrangement of the
F8 gene

kb C 2 3 4
21.5

16
14

The rearrangement changes the length of DNA

between restriction sites and hence the size of Genomic DNA digested with BclI, run on
fragments visualised on Southern blot agarose gel, transferred to filter &
Defect in Factor VIII or IX
In both cases it is an X-linked condition. hybridised with probe to F8A1. Different
Milder disease - residual function patterns depending on precise
Severe disease - little/no function
recombination event (F8A1 with F8A2 or
F8A3). C = normal control; 4 = patient
with point mutation (not the inversion).
 REFLP-family specific tracking
• They may or may not be associated with a diseases but used ONLY to track a
mutation in a specific family.
• Southern Blot can discriminate between alleles in an individual if a
mutation/polymorphism changes the size of the DNA fragments -
Restriction Fragment Length Polymorphism (RFLP)
• Bellow is an analysis of an X-linked trait
Sickle cell disease is due to a single
base change in the b-globin gene
Everybody with sickle cell disease is
homozygous for the same mutation

Straightforward, specific testing is available Probes hybridise

The sequences shown in blue & red are the only to completely
probes complimentary to either normal or complimentary
mutant sequences, respectively. DNA

Fig. 6.5 ©Scion Publishing Ltd

CGH or SNP array [Higher resolution] and multiple congenital Anomalies
Comparative Genomic
Hybridization
- A similar technique
but with a higher
resolution is to use
an SNP Array
DNA Sequencing & Nail Patella Syndrome
 Common types of DNA polymorphism
CA-rpt marker - a type of VNTR

Single nucleotide polymorphism (SNP)

Restriction fragment length polymorphism (RFLP)

RFLPs represent a sub-type of SNP

In family-based studies there has to be some way to tell
the chromosomes apart i.e. which is maternal or paternal,
which carries the mutant gene?
Gene Tracking

188, 200
58. Modes of Inheritance
Are there affecteds in
multiple generations?

YES NO

Are males & Autosomal

females affected Recessive
equally?

YES NO

More females More males

Is there male to affected than affected than
male transmission? males? females?

YES YES YES

Autosomal Dominant X-linked Dominant X-linked Recessive

 Pseudodominance and Complementation
• Pseudodominance: a recessive allele
is so common in the population that it
would be possible for a homozygote to
have children with a heterozygous
carrier e.g. ABO, blue eyes. Sickle cell
disease in parts of Africa, CF in part of
Quebec. See pedigree (redish)
• Complementation: Both parents have
the same recessive phenotype but all
kids are unaffected because they are
heterozygous at both loci.
 What is the chance that Judith’s sister is a carrier? (1/2)

What is the chance that Martin’s female cousin will have a

child with DMD?

½ x ½ x ¼ = 1/16
Chance that Judith’s Chance that she Chance that a carrier has
sister is a carrier passes mutation to an affected child
daughter
 Mendel’s Laws of Inheritance
• Uniformity - crossing two
homozygotes with different
alleles yields identical F1
offspring - all heterozygous

• Segregation - each individual

carries two genes for a
characteristic but only one is
passed on to the next
generation at each mating

• Independent assortment -
members of
• different gene pairs segregate
to
• offspring independently of each
other
• (this law is broken by linkage)
59. Genetics Terms
Codominance:
• Both alleles contribute to the phenotype of the
heterozygote
• Blood groups: AB
• α1-antitrypsin deficiency
• Mutations in the SERPINA1 gene cause alpha-1 antitrypsin
deficiency. This gene provides instructions for making a protein
called alpha-1 antitrypsin
• autosomal codominant pattern of inheritance
Linkage Disequilibrium:
• tendency for certain alleles to occur together more
than expected. This tendency is measure relative to a
specific population and not a specific family.
Penetrance: The proportion of individuals with a mutation
who exhibit the phenotype
Property of both gene & phenotype
- BRCA1 gene mutation

Variable expressivity: Different symptoms (or severity of

symptoms) in individuals with same mutation/genotype
- NPS
- NF1

Genes (& their products) act together

Pleiotropy
• One gene contribute to multiple phenotypic effects
• Untreated PKU results in light skin, intellectual disability,
and musty body odor
• Sickle cell disease
Loss of Heterozygosity
• If a Pt inherits or develops a mutation in a tumor
suppressor gene. The complementary allel must be
deleted /mutated before cancer develops.
• Incorrect for oncogenes.
• Retinoblastoma
• “two-hit hypothesis
 Mosaicism
• XIST mechanism of random X-
inactivation.
• PAR region on the X-chromosome is not
inactivated
• All females are mosaic.
• XIST RNA coats Xi and essentially turns Xi
into heterochromatin - except the
pseudoautosomal region - DNA
methylation, histone modification,
chromosomal structural proteins etc.
• Initial inactivation is a 2 step process with
Xist binding to gene-rich regions first. The
return of Xist after mitosis is a more rapid
single step process.
 Why it is important to know the mode of inheritance

Mosaic - composed of two different cell populations e.g. one with a

mutation, one w/o. It could be the result of a mitotic error after fertilization.
This results in genetically different cells in one individuals.
- Somatic Mosaicism
- Gonadal Mosaicism
SOMATIC mutation after fertilization - if in parental germline, can be
passed on (c.f. cancer, mutations in somatic tissue)
Mosaicism-FRAXA

Since the inactive X is

methylated there are
effects on diagnosis of
X-linked disease
Southern blot analysis
of the FMR1 gene in
FRAXA family
Skewed X-inactivation PAR
Genes that
sometimes/often

• DMD case= skewed

escape inactivation
Genes that don’t
escape inactivation
selection
Selection against lymphocytes in which the mutation is on the
active X gives the appearance of skewed inactivation
X-linked sever combined immunodeficiency (X-SCID)
CAG repeat laboratory techniques integration
IL2RG gene is required in B Cell development

Tracking
inactivation
using the CAG
rpt in AR gene.
HpaII wont cut
methylated
DNA

Fig. 7.13 ©Scion Publishing Ltd

Uniparental Disomy (UPD) Via trisomy rescue
• Two types:
• Isodisomy: Uniparental isodisomy can also result in
homozygosity for a recessive allele with only one
heterozygous parent. Nondisjunction Meiosis II
• Heterodisomy: nondisjunction in meiosis I
• UPD of an Entire Genome-Clinical Relevance
• Hyadatiform Mole there is complete and partial
• Ovarian teratoma which is a complete maternal
disomy
 60. Hardy-Weinberg population gene
• Factors that disrupt a H-W p2 + 2pq + q2 = 1
equilibrium p = f(1st allele)
• In an X-linked recessive disease q = f(2nd allele)
q= males and female=q2 p+q=1
Assumes:
• Heterozygote Advantage Random mating
No natural selection
• Malaria-sickle cell disease No mutation
No net migration (gene flow)
• Starvation- Non-insulin No genetic drift due to a small
dependent diabetes population size
Calculations to consider

What is the chance that Joanne’s brother is a

carrier?
David’s sister is thinking of having more children -
what is the chance her next child will have CF? (the
population incidence of CF is 1:2,500) The chance
that David’s sister’s next child will have CF is the
chance that she is a carrier (1/2), the chance that
her husband is a carrier (from H-W: √(1/2,500) x 2 =
1/25), the chance they both pass on the mutant
allele: ½ x 1/25 x ¼ = 1/200
 Exposure of alleles to selection

X-linked conditions - 1/3 X chromosomes are in males, for genetically lethal

conditions (e.g. DMD) 1/3 mutations are lost each generation so there must
be an appreciable number of new mutations - 2/3 risk mother of affected is
carrier w/o a family history

Fig. 10.5 ©Scion Publishing Ltd

 *

What is the chance that Judith’s sister is a carrier? ½

What is the chance that Martin’s female cousin (*) will have
a child with DMD?
½ x ½ x ¼ = 1/16
 *

What is the chance that Judith is a carrier?

What is the chance that Martin’s sister (*) will have a child
with DMD?
⅓ x ¼ = 1/12

Now what’s the chance Martin’s nephew has DMD?

⅓x½=⅙
• Autosomal Dominant Glomus Tumors inherited from father
61. Imprinting: Molecular Basis
• Beck-Weideman Syndrome (BWS) inherited from mother
• Proposed mechanism by which DNA methylation leads to
transcriptional repression. (a) Transcriptionally active chromatin
is predominantly unmethylated and has high levels of acetylated
histone tails (short black squiggles). (b) Methylation at CpG
dinucleotides can be carried out by one of the three known
human DNA methyltransferases (DNMT1, 3a and 3b), resulting in
DNA with high levels of CpG methylation (purple circles), but still
containing predominantly acetylated histone tails. DNA in this
form would still be expected to be transcriptionally competent. (c)
Methylated DNA is targeted by methyl-binding domain (MBD)
proteins such as MBD2 and MeCP2, which are found associated
with large protein complexes such as the NuRD complex (MBD2)
and the Sin3a complex (MeCP2). Histone deacetylase (HDAC1 and
2) and chromatin-remodelling activities (Mi-2 and Sin3a) within
these complexes result in alterations in chromatin structure,
producing chromatin that is refractory to transcriptional activation
(pink streaks represent deacetylated histone tails). The functional
roles of other components in these complexes are not yet known.
Abbreviations: MTA2, metastasis-associated protein 2;
RbAp46/48, retinoblastoma-associated protein 46/48; RNA pol II,
RNA polymerase II; SAP18/30, Sin3-associated polypeptides 18/30
Imprint Re-establishment in gametogenesis
- Some genes are expressed only from the paternal allele (maternally imprinted) and vice versa.
- Genes are not inherently paternal or maternal – the term only refers to whether they were most recently in
sperm or eggs.
- The epigenetic imprint is re-set every time a gene passes through gametogenesis.
Classical Example
• Prader-willi syndrome: maternal
imprinting
• Parental defect/deletion
• Hyperphagia
• Obesity
• Intellectual disability
• Hypogonadism
• Hypotonia
• Angelman syndrome: paternal
imprinting
• Maternal defect/deletion
• Happy puppet= inappropriate
laughter
• Seizure
• Ataxia
• Sever intellectual disability

Recent evidence suggests that

absence of an snoRNA, HBII-85,
may be the cause of PWS
62. Autosomal Dominant (AD) Diseases:
ADPKD (Autosomal Dominant Polycystic Kidney Disease)

• Bilateral enlargements of the

kidneys due to multiple large
cysts.
• 85 % of Pts have a mutation in
PKD1 gene of chromosome 16.
• 15% of Pts have a mutation in
PKD2 gene of chromosome 4.
• There is an autosomal
recessive version of the disease
called ARPKD
Haploinsufficiency

• This is a loss of function mutation. Could be due to:

Missense, nonsense, frameshift, deletion,
translocation…etc. All mutations in the same gene
result in the same phenotype.
• 50% is not enough resulting in a phenotype.
• Allelic Heterogeneity is a common feature
Nail Patella Syndrome
• This is a good classical example of
pleiotropy and variable expressivity
and Allelic Heterogeneity.
• Mutations in transcription factors
often cause a range of clinical
features because the same factor is
used in different cell types at
different stages in development.
Note that most, not all, mutations
act via haploinsufficiency. Such as
LMX1B
Disorders of Porphyrin Metabolism
• Acute Intermittent
Porphyria (AIP)
• Haploinsuficency of
Porophobilinogen
Deaminase
• Not photosensitive
• Variegate Porphyria
(VP)
• Haploinsufficiency in
Protoporophringen
Oxidase
• Photosensitive (see skin
bellow)
Gain of Function- FGFR3 Spectrum
• Achondroplasia
• Autosomal dominant, 80% cases are sporadic, ~1:20,000 live
births, rhizomelic (proximal) shortening of long bones,
depressed nasal bridge, pronounced lumbar lordosis,
p.G380R mutation
• Hyperchondroplasia (HCH)
• Thanatophoric Dsplasia
• Thanatophoric = “death loving”, always sporadic, ~1:20,000
births (usually stillborn), micromelic limb shortening, severe
frontal bossing, v. small chest
• TD-I - curved femurs, +/- kleeblattschaedel (cloverleaf skull)
• TD-II - straight femurs w/ kleeblattschaedel

• Apert Syndrome=FGFR2 is expressed predominantly in the

developing skull so mutations result primarily in skull defects
with minor limb defects
• These are gain of function where we have an up regulation or novel
function due to a missense mutation.
• Different mutations in the same gene result in different
phenotypes
Experimental Testing

1. Knock out the gene – if we see

the same phenotype(s) in ko mice
then must be haploinsufficiency.
2. “Knock in” mice – if the
phenotype is mutation-specific
then we are looking at gain of
function.
Spider Lamb Syndrome and Spider Leg Syndrome

• Spider Lamb Syndrome:

Loss of function p.V700Q
mutation in tyrosine
kinase domain of FGFR3
• Homozygosity for
p.T546K in FGFR3 results
in tall stature, severe
lateral tibial deviation,
scoliosis & campodactyly
(spider leg syndrome)
 Fibrodysplasia Ossificans Progressiva (FOP)
Congenital malformation of big toes
Progressive heterotopic ossification of skeletal muscles,
tendons, ligaments and fascia
Characteristic progression
- dorsal ventral
- axial appendicular
- cranial caudal
- proximal distal
Rare, autosomal dominant, most cases sporadic
R206H see the protein model image

BMP/TGF-β pathway
 Dominant Negative Mutation
• Osteogenesis Imperfecta
Type II and III
• OI Type II this is neonatal
lethal with multiple in utero
fractures.
• Dominant negative glycine
substitution.
• Dominant Negative is
where a mutant protein
interferes with a normal
copy.
• Missense or splicing
mutation and mostly refers
to structural proteins
Stickler Syndrome

• Due to Type II collagen null mutation or associated

collagens.
• Retinal detachment may occur
• Haploinsufficency
 Polydacylyl and the SHH gene
• Linkage studies in a large family
segregating polydactyly as an AD
characteristic suggested a mutation
in or near the SHH gene.
• Mutations of SHH are one cause of
holoprosencephaly in humans -
these polydactyly patients do not
have SHH mutations.A mouse
polydactyly mutant mapped to the
same region - found to be an
insertion w/i an intron of the gene
upstream from SHH. Insertion
disrupts a region that is highly
conserved during mammalian
evolution
• This conserved region functions as an
enhancer of SHH in the distal limb.
Enhancer mutation far upstream of
SHH results in polydactyly and not
holoprosencephaly
• Different enhancers can direct the
expression of the same gene in
different tissues
Lobar HPE and Campomelic Dysplasia
• Pt with lobar HPE there is
a single bp mutation in
the enhancing region
controlling SHH
expression during brain
development.
• SBE2= SHH brain enhancer
• ZRS= ZPA Enhancer limb
development enhancer
• Campomelic Dysplasia= is
an example of a distant
chromosomal mutation
disrupting gene
expression
Lactose Intolerance
• Is the inability to
metabolize lactose because
lactase is not expressed.
• Other variants in this region
associated with LP in other
populations exhibiting LP -
suggests selection for
different variants as dairy
production developed at
different times in human
history
• LP is an AD disease
Familial Adenomatous Polyposis
• Onset= after puberty
• Could evolve into colon
cancer
• APC gene on chromosome
5q is mutated
• AD disease

Micrograph of a tubular adenoma,

the colorectal cancer precursor most
commonly associated with FAP
Hereditary Hemorrhagic Telangiectasia
• Blood vessel disorders.
• Branching skin lesions [telangiectasia]
• Recurrent epistaxis
• Skin discoloration
• AVMs
• An AVM can develop anywhere in your
body but occurs most often in the brain CT-scan of vascular
or spine. A brain AVM, which appears as malformations in the liver in a
a tangle of abnormal arteries and veins,
can occur in any part of your brain. The patient with hereditary
cause isn't clear. hemorrhagic telangiectasia
causing an inhomogeneous
• GI bleeding perfusion pattern.
• Hematuria
The disease is also goes by
Osler-Weber-Rendu Syndrome Port swine stain
Mutiple Endocrine Neoplasia (MEN)
• It has subtypes such as
MEN1, MEN2A, MEN2B.
• Familial tumors of
endocrine gland,
including those of the
pancreases, parathyroid,
pituitary, thyroid, and
adrenal medulla.
• MEN 1 is due to a
mutation in MEN1 gene
• MEN 2A and 2B is due to
a mutation in RET gene
Von Recklinghausen Disease
[Neurofibromatosis type 1]
• NF1 gene onChromosome 17 is
mutated
• Variable expressivity but 100%
penetrance
• Neurocutanous disorder
characterized by:
• café-au-lait spots
• Cutanous Neurofibroma
• Optic gliomas
• Phenochromocytomas
• is a rare tumor of adrenal gland tissue.
It results in the release of too much
epinephrine and norepinephrine,
hormones that control heart rate,
metabolism, and blood pressure
• Lisch nodules= pigmented iris
hamartomas
Neurofibromatosis type 2
• Mutation in NF2 gene on
Chromosome 22.
• Results:
• Bilateral acoustic
schwannomas
• aka vestibular schwannoma
• Juvenile cataracts
• Meningiomas
• Ependymomas
• thin epithelium-like lining of
the ventricular system
Tuberous Sclerosis
• Neurocutanous disorder
with multi-organ system
involvement.
• Many benign
hamartomas.
• Incomplete penetrance
and variable expressivity. 70% of pts will have multiple
angiomyolipomas of the
kidney
Von Hippel-Lindau Disease
• Chromosome 3p
specifically a deletion in
VHL gene [tumor
suppressor].
• Pt will develop many
tumors of both benign
and malignant nature.

Typical distribution of
hemangioblastomas in
Von Hippel–Lindau
disease.
 63. Autosomal Recessive (AR) Diseases

Amino Acid Metabolism Disorders

Phenylketonuria, alkaptonuria,
albinism
Phe, Tyr metabolism

Homocysteinuria
Met Cys
Note the
Maple Syrup Urine Disease
Leu, Ile, Val metabolism DOPA
addition/removal
of moieties at
each step
 PKU
• Phenylalanine hyroxylase deficiency. Results in
toxic effects of phenylpyruvate and other
metabolites. It is Autosomal recessive.
• Treat with low-Phe diet even during pregnancy.
If PKU mother does not stick to a low-Phe diet
while pregnant, child will be mentally retarded
despite an Aa genotype
• Allelic Heterogeneity
 Heterogeneity
• Locus: Mutations in different genes result in the same
pheontypes
– Albinism
– Osteogenesis Imperfecta COL1A1 or COL1A2
– Hereditary Spherocytosis
• Allelic: different mutations in the same gene cause
the same disease
– β-Thalassemia
– NPS
• Genetic heterogeneity includes both locus and allelic.
Hyperphenylalanaemia
A small percentage of cases of
hyperphenylalanaemia are due to deficiency
of the co-factor tetrahydrobiopterin (BH4). A
low-Phe diet will not prevent MR in these
patients since BH4 is also required for
neurotransmitter synthesis.
Locus heterogeneity
 Alkaptonuria Homogentistic acid
oxidase deficiency
Autosomal recessive
Accumulation of
homogentistic acid
1:250,000 - 1:1,000,000
A. Bluish-black discolouration of
the sclera.
B. Bluish-black discolouration of
the auriculum.
C. Following standing, the
patient's urine blackened.
D. X-ray (left anterior oblique
45°) of the aortic valve revealed
severe calcification.
E & F. Extensive calcification and
bluish-black discolouration of the
tricuspid aortic valve and
discolouration of the aortic intima
were found on operation.

http://eurheartj.oxfordjournals.org/content/vol29/issue4/cover.dtl
 Oculocutanous Albinism
• OCA1- tyrosinase deficiency. Where the inability to convert
tyrosine melanin
– Lack of eye pigment affects visual acuity and projection of
visual pathways to the optic cortex
– Locus heterogeneity.
• An enhancer mutation is the cause of blue/brown eye color
variation
– single base change w/i intron of gene upstream from OCA2
– OCA2 gene involved in production of melanin via P protein
(unknown function as of now)
• See imprinting example bellow
 Homocystinuria
Deficiency of cystathionine b-synthetase (CBS)
Mental retardation
Fits
Osteoporosis
Low Met diet helps
Methionine
Autosomal recessive

Homocysteine
CBS
Cystathione

Cysteine
 Maple Syrup Urine Disease

Deficiency of branched-chain ketoacid

decarboxylase
Excretion of Val, Leu, Ile
Fatal if untreated
Overall incidence of 1/180,000 but >
1/200 in PA Mennonites
Diffuse hypoattenuation of
Autosomal recessive
cerebral and cerebellar
white matter
Haemoglobinopathies
Development and Chromosomal Location

• KNOW the
composition of
embryonic (ζ2ε2),
foetal (α2γ2) and
adult (A & A2;
α2β2 & α2δ2 )
haemoglobin.
Thalassaemias
• Hydrops Foetalis= Hb Barts, γ4
• No a-globin synthesis severe anaemia
heart failure oedema
• Hb H
– Milder than Hb Barts some a-globin
synthesis. majority of Hb is b-tetramer (b4,
unstable)
• Hb Constant Spring 4, b 4 and CS have O2 affinities
comparable to myoglobin => no
– This is phenotypically similar to Hb H but
release to tissues
mutation of stop codon in a-globin gene
leads to longer, unstable protein
• α-thalassaemias- due to unequal crossover
– Alph+ thal is a mild form of alpha-thal
• β-thalassaemias [cooley’s anaemia] is due to
mutations in the Betta-hemoglobin gene.
– Mostly a promoter TATA box mutation
– But missense mutations cause sickle cell Silent
anemia for example carrier=not
– Enlargement of cheek bones and maxilla. affected
Distinct abdominal swelling from
hepatosplenomegaly.

Note that in Hb-Lepore, transcription is from the -globin promoter so the

hybrid gene is expressed at the same low level as (~2% of b). Hb-Lepore is
a b-thalassaemia allele, anti-Lepore is essentially normal.
HPFH= Hereditary Persistence of Foetal Haemoglobin
Deletion of - & b- genes removes promoter elements that LCR would interact with
- no competition for -gene promoter so chain production continues post-natally
Normal phenotype unless exposed to low O2 Also, pregnant will not be able to pass
Hb Leopard to their baby oxygen due to impaired diffusion.
- Also, due to a mutation in silencer elements of the -gene.
 Special Clinical Case and
nomenclature
• Remember that a- thalassaemia is
the result of gene deletion but that b-
thalassaemia is almost always due
to null point mutations
• b-thalassaemia: b+-thal - trace b-
globin synthesis (leaky mutations).
b0-thal - no b-globin synthesis (major
mutations).
• A child was diagnosed with b+-
thalassaemia but appeared milder
Status Relative a chains Relative b chains
than typical cases. Further
investigation confirmed b+-thal but Normal 100 100
also showed him to be heterozygous b+-thal 100 10
for a-thalassaemia (a+/-). a+/- 50 100
– Compare the relative excess of a+/- b +-thal 50 10
a chains – less a chain excess,
fewer a4 tetramers, less lysis
 64. Trinucleotide Repeat Expansion Diseases
• Anticipation: is the increased severity/earlier onset of a disease in succeeding generations.
• Huntington disease: results in chromosome 4 and causes depression, progressive dementia, choreiform movement, and
caudate atrophy.
• Increase dopamine, and decrease in GABA and Ach in brain. Death of motor neurons
• Huntingtin (HTT) protein defect
• Paternal expansion bias: Characteristic of most CAG expansion (poly Glutamine) disorders, such as
Huntington and the cerebellar ataxia’s (SCA)
• Maternal expansion bias: Characteristic for the CGG expansion in the Fragile X syndrome (FRAXA) and the
CTG expansions in Myotonic Dystrophy (DM1)
Diseases that are caused by loss of protein (null mutations): Fragile X syndrome (FRAXA, X-linked
dominant); Friedreich Ataxia (FRDA, autosomal recessive)
Diseases that are caused by altered RNA function (gain of function mutation): Myotonic Dystrophy I (DM1,
autosomal dominant); Myotonic Dystrophy II (DM2, autosomal dominant); Fragile X Tremor Ataxia Syndrome
(FXTAS, only in older males)
Diseases that are caused by altered protein function (gain of function mutations); Huntington (HD,
autosomal dominant); Spinocerebellar ataxias (SCAs, autosomal dominant)
AUG TAA
5’ 3’

(GAA)n
(CGG)n Friedreich (CAG)n (CTG)n
Fragile X
syndrome
Ataxia - Huntington Myotonic
- SCA
- others
Dystrophy
Fragile X Syndrome (FRAXA)
• X-linked dominant defect affecting the In proliferating cells, triplet repeat instability is
methylation and expression of FMR1 gene. provoked during processing of Okazaki fragments
consisting of trinucleotide repeats. Looping out
• It is the 2nd most common cause of genetic of the Okazaki fragments gives expansions and
intellectual disability after Down Syndrome
looping out of the template strand gives
• A trinucleotide repeat syndrome due to CGG contractions. For resolution or repair of the
repeats. looped out structure, recognition by RPA,
• Result: unwinding by proteins that have helicase and
• Post-pubertal macroorchidism (enlargement of nuclease activity (FEN-1, Dna2) and DNA
testes). polymerases are needed.
• Long face with a large jaw
• Large everted ears In quiescent cells the mismatch repair genes
• Autism Msh2 and Msh3 recognize the looped out
• Mitral valve prolapse structure and then these structures are
• Occurs more profoundly in males compared to
females where it is even less penetrant (only 50% of processed by the same proteins involved in
heterozygous females will have FRAXA) triplet repeat instability in proliferating cells.
• Occurs on the Xq arm and less than 1% will be due to
point mutations [missense] /genetic deletions. FXTAS= neurodegeneration in older male
• Normal transmitting males (NTM): have the
permutation but not the disease/fragile site. where there is an in crease of FMR1
• The premutation is also associated with an increased mRNA with reduced trn. Histologically we
risk of disorders called fragile X-associated primary
ovarian insufficiency (FXPOI) and fragile X-associated will observe inclusions in FMR1 mRNA
tremor/ataxia syndrome (FXTAS).
• The FMR1 gene is silenced via CGG methylation and
makes very little or no protein. A loss or shortage of
FMRP disrupts normal functions of nerve cells and,
consequently, the nervous system, causing severe
learning problems, intellectual disability, and the
other features of fragile X syndrome
 Friedreich Ataxia [FRDA]

• Life shortening and neurodegenerative disorder associated with weakness in muscles and
hearing.
• AR and shorter rpt= better
• NO ANTICIPATION!!!!
• Frataxin deficiency
• 2% of cases due to point mutation [allelic heterogeneity]
• Excess iron in mitochondria because frataxin is needed for haem biosynthesis and formation
of Fe-S clusters
• Reduced function of respiratory chain complexes I-III and aconitase: energy deficiency and
excess free radical formation
 Mytonic Dystrophy 1 (DM1)
• This is cause by an expansion in the 3’ UTR of the DMPK gene on
chromosome 19q. The repeat titrates out the important splicing proteins.
• Rsults in myotonia due to chloride channel 1 defect [CLCN1]; cardiac abnormality due
to defect processing [cardiac troponin T]; memory defect due to [APP]
• Gain of function mutation
• Myotonia, cataract, hypogonadism, and frontal balding
• AD multisystem disorder
Spinocerebral Ataxia [SCA]
• Degeneration of cerebellum both afferent and efferent
connections with brainstem and spinal cord.
• progressive problems with movement especially
coordination and balance
• AD disorder. Mutations in the ATXN1 gene cause SCA1.
The ATXN1 gene provides instructions for making a
protein called ataxin-1
• This protein is found in the nucleus.
• Involves exonic CAG repeats.
• Gain of function
• 25 different possible loci
 Pathogenesis of polyglutamine diseases

• Different neurological diseases arise not so much because

of specific expression of the genes involved, but because
specific neuron populations are vulnerable
• Central pathological feature of polyglutamine diseases:
insoluble aggregates or inclusions
• Current evidence: inclusions are protective, in the
vulnerable cell populations no inclusions are seen
• Nuclear localization of the mutant protein is crucial for
pathogenesis > global transcriptional changes precede
cellular phenotypic onset
 Multiple
pathogenic
mechanisms for
polyQ expansions

Cleavage
Aberrant interactions
Aggregates
Transcription
dysregulation

Histone deacetylation
& methylation a major
mechanism of mHTT
action
Htt is expressed in all cells
- why is the pathology so specific?

Mutant Htt (mHtt) aggregates in cells

- probably protective, aggregation doesn’t kill cells

mHtt is modified by SUMO et al - sumolyation keeps mHtt

soluble - cytotoxic

aggregate soluble

Rhes binds mHtt moreso than Htt and promotes sumolyation

Rhes is expressed specifically in the striatum and at lower

amounts in the cerebral cortex - hence striatal phenotype
(Rhes = Ras homologue expressed in striatum)

btw, ataxin does not bind Rhes.

65. Trisomies
• Down Syndrome (21): intellectual disability, flat
face, prominaent epicancanthal folds, single palmar
crease, gap between 1st and 2nd toes, duodenal
atresia, Hirschsprug disease.
• 95% complete trisomy
• 4% Robertsonian Translocation
• 1% Mosaic due to a nondisjunction during mitosis
• Edwards Syndrome (18)
• Patau Syndrome (13)
Abnormal Chromosome Number
• Turner Syndrome= 45,X [see next slide]
• Klinefelter syndrome
• Characterised by female secondary sex characteristics,
including gynaecomastia (breast development) and a female
distribution of pubic hair. The legs are disproportionately
long.
• 47XXY karyotype
• 1/500 males
• Long limbs
• IQ ~85-90
• Hypogonadism
• Treat with testosterone supplementation
Clinical features and karyotypes of Turner syndrome

~57%
~14%

~30%

FISH & PCR studies suggest ~75% of

cases are mosaic - theory that no
liveborn is truely 45X

Greater proportion of 45X cells

associated with more severe phenotype

~50% of cases develop thyroid

autoantibodies

Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 13 June 2007 02:16 PM) © 2007 Elsevier
 66. Robertsonian Translocation
• Results from the
recombination between
the rDNA sequences on
non-homologous
acrocentric
chromosomes. Such as
13, 14, 15, 21, and 22.
Types of chromosomes and
Robertsonian Translocation Outcome
67. Cri-du-chat syndrome
• Microdeletion of the short arm in chromosome5
[46,XX or XY,5p-].
• Microcephaly
• Intellectual disability
• High pitch crying/mewing
• Epicanthal folds
• VSDs are common
 68. Williams Syndrome
• Congenital microdeletion of the long arm of chromosome 7. The
deletion includes an elastin gene.
• Elfin face
• Intellectual diasability
• Hypercalciemia due to increase sensitivity to vitamin D.
• Good verbal skills
• Very very very friendly to strangers.
• Cardiovasucal problems

Refer to subsequent figure in next slide

Figure 3. The Williams-Beuren Syndrome Chromosome Region (WBSCR) on Chromosome 7. Panel A (top) shows the WBSCR located
between flanking blocks of low-copy DNA repeats, known as duplicons. Panel A (middle) also shows the most common WBSCR
deletions, approximately 1.5 Mb and 1.8 Mb in size; breakpoint regions responsible for these deletions occur in the centromeric and
medial duplicon B blocks and the A blocks, respectively. An enlargement of the WBSCR, depicting the genes unique to this interval, is
shown in Panel A (bottom, with the width of the rectangle roughly corresponding to gene size). Panel A (bottom) also shows an example
of WBSCR deletions of atypical size. Cen denotes centromere, and Tel telomere. Panel B shows normal pairing of the two copies of the
WBSCR during meiosis, caused by alignment of the centromeric, medial, and telomeric duplicons on the chromosome 7 homologues.
Panel C shows abnormal pairing of the two copies of the WBSCR during meiosis, caused by misalignment of the centromeric and medial
duplicons due to their partial homology. Crossing over can result in abnormal recombinant products, either deletion of the WBSCR
(causing Williams-Beuren syndrome) or duplication of WBSCR. The open circles in Panels B and C denote the centromere. Panels A, B,
and C are schematized, not drawn to scale.
Elastin – heterozygous loss of function mutations in the gene coding for elastin (ELN) result in supravalvular aortic stenosis (SVAS). A BAC
probe for ELN is commonly used in FISH diagnosis of WBS.
 Deletion and Duplication of 7q11.23 Result
in Contrasting Phenotypes
Duplication Deletion
Deletion
Short philtrum Long philtrum
Thin lips Full lips
Larger teeth Small teeth
Arched palate Normal palate
Narrow forehead Broad forehead
Normal periorbital Periorbital
area fullness

Severe expressive Relative strength in

language delay expressive language

Relative strength in Very weak spatial skills

spatial skills
 The Williams-Beuren
Syndrome Chromosome
Region (WBSCR) on
Chromosome 7
Classic example of
contiguous gene deletion
syndrome

Normal alignment &

recombination

Recombination after
misalignment
For every deletion there should be a
corresponding duplication

Genes Rpts

Genes

Genes Genes

Recombination between flanking repeats results in one gamete

deleted for intervening sequence and one duplicated for same.
Deletions seem to result in a phenotype more often (c.f. monosomy vs
trisomy)
Deletion and duplication phenotypes may be approx opposite of each
other
69. 22q11 deletions
• Microdeletion in 22q11 variable presentation such as:
• Cleft plate
• Abnormal facies
• Thymic aplasia leading to T-cell deficiency
• Cardiac defects
• Hypocalcemia secondary to parathyroid aplasia
1) DiGeoge Syndrome: thymic, parathyroid, and cardiac
defects
2) Velocardiofacial syndrome: palatal, facial, and cardiac
defects.
• The defects are due to aberrant development of 3rd and 4th
brachial pouches.
70. X and Y Chromsomes
• X-linked Recessive Diseases
• Burton Agammaglobulinemia
• Wiskott-Aldrich Syndrome
• Fabry Disease
• G6PD deficicy
• Ocular albinism
• Leshch-Nyhan Syndrome
• Duchenne (and Becker) muscular dystrophies
• Hunter Syndrome
• Hemophilia A and B
• Ornithine Transcarbamylase deficiency
• Female carriers can be variably affected depending on
the percentage of inactive X-chromosomes containing
the mutant vs normal X.
SRY gene and Androgen Insensitivity/Testicular Feminization (TFM)
• SRY gene
expression=male. Thus,
it is possible to have ab
XY female and an XX
male (if there was an
SRY gene transferred).
• SOX9 and WNT4 are
expressed in the
gonadal ridge.
• TFM is an X-linked
mutation of the
androgen receptor
having a reduced
response to
testosterone.
• Sex differentiaition
Androgen Receptor (AR) gene
Alpha Thalassaemia with Mental Retardation - X-linked (ATRX)

Result of mutation in a SWI2/SNF protein

- a helicase that determines the local structure
of chromatin and hence regulates gene
expression. What do you expect to happen in
a heterozygote vs. a hemizygote?
- see text pp. 51-52 for details
- this is another example of selection
CFNS and the EFNB1 gene
• Unusual X-linked dominant condition.
• Severe hypertelorism
• Coronal synostosis
• Grooved tip of nose
• Longitudinal nail split
• This is an unusual X-linked dominant
condition in which males are less
affected than females. It emphasises
the fact that females are mosaic – two
different cell populations, and
sometimes that causes problems!!
• Loss of function mutation in EFNB1 a
transmembrane ligand for the Eph
receptor kinase
• It is expressed in the frontonasal neural
crest [NC] cells
• Marks the boundary between NC and
Mesoderm derived cranial mesenchyme
• CFNS results from imprecise boundry
forming due to random X-inactivation
• See receptor-ligand interaction at the NC-
mesoderm border in the illustration to the
right
 Rett syndrome
X-linked dominant
Leading cause of
profound mental
retardation in females
Normal development
until 6-18mo followed by
progressive loss of
milestones

Characteristic hand-ringing
Progressive microcephaly, ~50% seizures
Mutation in MECP2 (see later)
Occasional male patients usually present with severe
neonatal encephalopathy
Is regarded as the severe end of the autism spectrum
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=rett
71. DNA Mutations
• Null Mutation= no
protein product made
• Splice Mutations
• Cryptic Splice Site
• Indel Mutations
• Genes and Modes of
inheritance
• Recessive: Enzymes and
Transporters
• Dominant: Structural
proteins and TFs
Smith Magenis Syndrome
Distinctive facial appearance, Broad square-shaped face,
Brachycephaly, Prominent forehead, Synophrys, Upslanting
palpebral fissures Deep set eyes, Broad nasal bridge,
Midface hypoplasia, Short full tipped nose Micrognathia
progressing to prognathia with age, Distinctive mouth with
tented appearance to upper lip.
Hypotonia, Short stature, Brachydactyly, Eye and Ear
abnormalities, Speech delay, Scoliosis, Moderate mental
retardation, occasional cardiac and renal abnormalities
Etiology - Deletion 17p11.2 3700 kb
duplication causes Potocki-Lupski syndrome (PLS)
• NOTE: same mechanism of action as WBS
 Mode of inheritance?

Recessive Dominant

Loss of Null alleles observed?

function
mutations Yes No

Haploinsufficiency Do different missense

mutations yield distinct
phenotypes?

No* Yes

Dominant-negative Gain of function

* If null mutations are observed in the same gene and result in a milder phenotype, missense
mutations probably dominant-negative e.g. collagen
72. Congenital Adrenal Hyperplasia
• Steroid 21-hydroxylase deficiency
• AR disorder
• Virilized external female genitalia
• Male baby with hypospadias who clearly has testes in the scrotal sacs
• Lack of steroid (aldosterone) product impairs negative feedback control of adrenocorticotropin (ACTH) secretion from the
pituitary salt wasting
• Accumulated precursors shunted through uninhibited pathways excessive adrenal androgen biosynthesis virilization in all
individuals and in some salt wasting
• Impaired pituitary feedback chronic stimulation of adrenal cortex by ACTH adrenal hyperplasia.
• Gene Conversion is the mechanism.
73. Albright Hereditary Osteodystrophy (AHO) and PHP/PPHP
• AHO results in a short stature,
brachydactyly, and obesity.
• It is a AD disorder from a loss of function
mutation in the GPCR that activated
adenylyl cyclase.
• Pseudohypoparathyroidism (PHP)
- first human disorder of hormone resistance
(Albright, 1942)
PHP1a - AD, AHO + multiple hormone
resistance (PTH, TSH, LH, GHRH)
PHP1b - AD, renal PTH resistance w/o AHO
• Pseudopseudohypoparathyroidism (PPHP)
PPHP - AD, AHO w/o hormone resistance
• Maternal allele must be expressed in proximal
tubules for correct hormone response.
• In renal proximal tubules GαS is only
expressed from the MATERNAL allele. In renal
proximal tubules the use of exon 1A
PRECLUDES use of exon 1. Exon 1 must be
used to make GαS. Methylation of exon 1A on
the maternal allele prevents use of exon 1A
and therefore use of exon 1 and synthesis of
GαS . In other tissues the use of exon 1A does
not preclude the use of exon 1 so, even
though the same imprint is present, GαS is
expressed from BOTH alleles. Put this all
together – only a mutation on the MATERNAL
allele causes hormone resistance since only
the maternall allele synthesises GαS in these
cells.
74.McCune-Albright Syndrome
Somatic gain of function mutation in
GNAS

Polyostotic Fibrous dysplasia

Café au lait spots/patchs-unilaterally
Precocious puberty, hyperthyroidism,
hyperparathyroidism,
pituitary adenomas,
Acromegaly
Lethal if before fertilization
 75. Dyschromatosis Symmetirca
Hereditaria

Dyschromatosis symmetrica hereditaria

-hypo- & hyperpigmented macules on
dorsal aspect of hands & feet
- autosomal dominant
76. Recombination-Overview
• Recombination Hotspots: these are sex-
specific and they are where 95% of
recombination occurs.
• In pedigrees
• Genes far apart on the same chromosome
also exhibit 50% recombination since there
is most likely to be a recombination
between them
• The probability that alleles recombine
(cross-over) is related to the distance
between the loci.
• genetic distance
Linkage
• LOD score and its interpretation:
• Information on two loci (disease and marker locus) is collected in a group of families
• The likelihood of obtaining such data if the 2 loci are linked at a specific recombination frequency, , is
compared with the likelihood of their not being linked ( = 0.5)
• The ratio of the 2 likelihoods gives the odds for linkage (if > 1), or against linkage (if < 1)
• The ratio is converted to a log scale log of the odds = lod score
• LOD score of +3 is equivalent to a P=0.05 or less
• Lod score +3 or higher: evidence for the presence of linkage (likelihood ratio 1,000:1)
• Lod score -2 or lower: linkage is excluded (likelihood ratio 1:100)
• Lod score between -2 and +3: inconclusive
• Cumulative 2-point LOD scores
• Zmax needs to me more than or equal to 3 but also with a theta-max of the least possible value.
• Each marker is 60cM thus we need haplotyping for a higher resolution
Haplotyping Question
• Darier-White Disease= Autosomal dominant disease on chromosome 12.
Skin condition characterized by wart-like blemishes on the body. The
blemishes are usually yellowish in color, hard to the touch, mildly greasy,
and can emit a strong odor. Mutations in the ATP2A2 gene result in
insufficient amounts of functional SERCA2 enzyme. A lack of SERCA2
enzyme reduces calcium levels in the endoplasmic reticulum, causing it to
become dysfunctional.
• Taking all of the data together we can tell that the mutation is
between D1S2715 & D1S2777.
 Autozygosity Mapping

In a consanguinous recessive
pedigree, affecteds are homozygous
for the same mutant allele – and for
nearby marker alleles
Kabuki Syndrome and Exome
Sequencing
• Pt will have distinct facies and cardio-skeletal
defects. Immunological defects. Cases are sporadic;
Moderate retardation.
• The mutation will be narrowed to ONE GENE.
• MLL2 gene mutation with a dominant inheritance
mode.
• Thus, suspecting 1 gene with sporadic mutations of
non-related people having same phenotypes=
exome sequencing
 77. Founder Effect
• Tay-Sachs Disease
• Cherry-Red Spot on macula
• Progressive neurodegeneration
• Development delay
• Observe the ganglion with lipid
vacuolation
• NO hepatosplenomegaly
• Blood test= Hexoamindase A
deficiency with an accumulation o
GM2 ganglioside.
• AR inheritance found in Ashkenazi
Jews
• Gaucher Disease
• Type I where Enzyme Replacement The centre of the fovea appears bright red because it is
Therapy (ERT) works using a surrounded by a milky halo. This halo represents loss of
cerezyme. Also for bellow diseases: retinal transparency which comes from accumulation of
ganglioside in ganglion cells due to HexA deficiency. Since
• Fabry Disease an X-linked α- there are no ganglion cells in the centre of the fovea the
galactosidase A deficiency. underlying choroid transmits its red colour.
The centre of the fovea appears bright red because it is
• MPS I surrounded by a milky halo. This halo represents loss of
retinal transparency which comes from accumulation of
• Pompe Disease an α-glucosidase ganglioside in ganglion cells due to HexA deficiency. Since
deficiency. there are no ganglion cells in the centre of the fovea the
underlying choroid transmits its red colour. Vision is
subnormal because of a defect in the ganglion cells
78. Multifactorial Disorders
• Dichotomous vs Quantitative
• Dichotomous or discontinuous characters, e.g diseases
and malformations, that you either have or do not have.
• Continuous or quantitative characters, that everybody
has, but to different degrees, such as height, weight,
blood pressure.
• Multiple Sclerosis:
• used as an example where genetic factors play a major
part in determining susceptibility, but each individual
factor has a low penetrance.
 Modifier genes

SOD1 mutation ALS

CNTF affects age of onset
CNTF+/- unaffected w/o SOD1 mutation - hence it is a modifier

a | A CNTF-null allele modulates the age of

onset of the dominantly transmitted disease
amyotrophic lateral sclerosis (ALS).
Mutations in both SOD1 and CNTF lead to
early-onset ALS (age 25) and death within 11
months (the third-generation male in the + = normal allele
diagram). The third-generation female with a - = mutant allele
SOD1 mutation but no CNTF mutation did not
present with the disorder until the age of 54. b
| A pedigree of digenic inheritance showing
how retinitis pigmentosa occurs only in
individuals who have inherited one mutation
in each of ROM1 and RDS. Heterozygotes for
either mutant allele are asymptomatic. CNTF,
ciliary neurotrophic factor; RDS, retinal
degeneration slow; ROM1, retinal outer
segment membrane protein 1; SOD1,
superoxide dismutase 1. Circles indicate
females; squares indicate males; diagonal red
line across symbol indicates lethality.

Digenic inheritance

RP due to heterozygous mutations in BOTH ROM1 and

RDS. Individual heterozygotes are unaffected.
 Threshold and Liability Model &
Heritability
• When a condition is
only genetically
determined the H2 =
1
• When a condition is
only determined by
environmental
factors, the H2 = 0
• Many multifactorial
disorders have H2 in
the range of 0.4 – 0.7
Familial Aggregation
• Calculation of relative risk (RR):
• Familial relative risk in Mendelian disorders
• Example: Huntington’s disease (AD)
– 50% of siblings affected
– Population frequency 0.005% (1:20,000)
– RR = 10,000
– Conclusion: A sibling of a patient with HD is 10,000 times
more likely to be affected than a random individual
• Familial relative risk in multifactorial disorders
• Example: Type 2 diabetes mellitus (T2D)
– Number of T2D relatives exhibiting T2D: 18/100
– Number of relatives of controls exhibiting T2D: 5/100
– RR = (18/100)/(5/100) = 3.6
– Conclusion: A relative of a T2D patient is 3.6 times more
likely to be affected than a relative of a non-T2D individual
Twin and Adoption Studies
Concordance rate:
• Monogenic disorder
• MZ twins 100% concordance
• DZ twins 50% concordance
only genes involved
• Multifactorial disorders, e.g. T2D
• MZ twins ~ 80% concordance
• DZ twins ~ 40% concordance
genes play a significant role, but not solely!
Twin studies:
• Test: comparing the disease concordance rates among MZ (identical) twin pairs with those of DZ (fraternal) twin pairs
• MZ twins: share 100% of their genes
• DZ twins: share 50% of their genes
• If genetic factors play a significant role in disease etiology: MZ twins will have higher concordance rates than will DZ twins
• Bias in twin studies:
• MZ twins are more likely to be very similar, to be dressed and treated the same
• Share more of their environment than DZ twins
• Ideal experiment: MZ twins separated at birth and brought up in entirely separate environments.
Adoption Studies:
• Most powerful to disentangle genetic and environmental factors
• Test: to determine if individuals are more concordant with their adoptive parents or with their biologic parents for a disease
• more concordant with their adoptive parents common environment is responsible
• more concordant with their biologic parents genes are implicated in disease etiology
79. Non-Parametric Linkage Analysis

• Does not require knowledge on the parameters used in

lod score analysis = model-free analysis
• Mainly allele-sharing approaches
– Family-based: affected sibpair analysis
– Population-based: association studies
• Commonly used for multifactorial genetic models
80. Affected Sibpair Analysis
• Most common non-parametric
method of linkage analysis
• Underlying concept
• At a single genetic locus an
offspring can receive any 2 of 4
parental alleles
• Pairs of siblings can share 2, 1, or
0 alleles transmitted from their
parents
• Prior probabilities: Z2=0.25,
Z1=0.5, and Z0=0.25
• Linkage will cause a shift in this
distribution
Allele Sharing:
• Identical by descent (IBD)
• The same identical
allele (inherited from
the same parent)
• Identical by state (IBS)
• The same allele, but
not necessary identical
81. TDT= Transmission Disequilibrium Test
82 & 83.Linkage and Association
Studies
Case-control design:
Test for differences in
genetic marker
frequency between
affected cases and
unaffected controls
- Excess of certain
marker alleles in
affected cases indicates
association between
DNA marker and
disease
84. Age Related Macular Degeneration
• Age-related macular degeneration (AMD) - leading cause of age-
related blindness in developed world.
• Known to result from genetic and environmental factors.
• Linkage studies identify candidate regions but no causative
mutations.
• Whole-genome case-control association study:
• 96 cases, 50 controls
> 100,000 SNPs
rs380390 P = 0.0043 (uncorrected P = 4.15 x 10-8)
Part of a haplotype with a RR of AMD of 4.6 (c.i. 2-11) in
heterozygotes and 7.4 (c.i. 3-19) in homozygotes
85. Celiac Disease Twin studies
- Concordance MZ twins 86%
- The most common food intolerance in western populations (0.5- - Concordance DZ twins 20%
1%)
- Abnormal immune response to gluten proteins in wheat, barley -> Important role for
and rye environmental factors (gluten)
- Leads to inflammation and damage in the small intestine of
genetically susceptible individuals
- Results in malnutrition and associated complications Family studies
• >95% of all patients carry the HLA-DQ2 gene (6p25) - Increased familial clustering
>95% of CD patient express the HLA-DQ2 or HLA- - CD in general population ~ 0.5%
DQ8 heterodimer - CD in sibling of CD patient ~10%
BUT: -> Important role for genetic
factors
• ~25% of general population also expresses HLA-
DQ2/DQ8
• Only ~0.5-1% of general population develops CD!
• Concordance for monozygous twins much higher
than for HLA-DQ2/DQ8 matched controls
• Sibpair study revealed genetic loci on 6q21 and
19p13.1 (the gene is called CELIAC4, a minor gene)
• Myosin IXB variant encodes an unconventional
myosin molecule that has a role in actin remodelling
of epithelial enterocytes
86. Hirschsprung Disease
• Congenital intestinal aganglionosis (absence of nerve
cells in the large bowel, no bowel movement causing
abdominal distension)
• Defect in the migration of neural crest-derived
intestinal ganglion cells to the intestinal tract during
development
• Recurrence risk: 3-4% >RR= 200
• Sex ratio 4:1 :
• Proportion of affected sibs is higher when the index
patient is a female - why?
- Given the lower risk to , an affected must have more
bad genes so her relatives are also more likely to have
more bad genes
Genes involved in Hirschsprung Disease

System 1
• RET: Receptor tyrosine kinase. Promotes neural crest stem cell
migration to the intestine. Gene found by linkage analysis
Effect is dominant. Penetrance 50-70%. Loss of function
mutations. The gene most often associated with Hirschsprung
disease.

• RET ligands: GDNF (Glial cell line-derived neurotrophin factor)

and NRTN (neurturin). GDNF has a role in migration of neural
crest cells and NRTN promotes their survival. Genes found by
candidate gene analysis. Insufficient to cause Hirschsprung
disease by themselves.
i.e. GDNF & NRTN increase risk of Hirschsprung in presence of
a RET mutation.
Genes involved in Hirschsprung Disease
System 2
• EDNRB: Endothelin receptor kinase type B - found by
linkage analysis in large Mennonite family with
syndromic Hirschprung disease (associated with
sensineural deafness, white fore lock)
• EDN3: ligand for EDNRB
• Gene effects are recessive. Involved not only in spread
of neural precursors from the small bowel to the large
bowel, but also of melanocytes. Each by themselves
implicated in types of Waardenburg syndrome (white
forelock, deafness)
Genes involved in Hirschsprung Disease
System 2 continued
• SOX10: transcription factor, required for
differentiation of enteric glial cells. A potential site of
action of SOX10 is an enhancer element of the EDNRB
locus that is spatiotemporally activated at the time
that enteric nervous system precursors are populating
the distal gut. SOX10 gene mutations are found in
dominant HSCR syndromes.
• Mutations in RET, EDNRB, EDN3, GDNF and SOX10
account for 60-70% of familial cases of Hirschsprung
disease and 10-30% of the sporadic cases
 87. Diabetes
Type I:
• 2-3/1000 in 10-19 year age group
• Autoimmune disorder: destruction of pancreatic islet beta cells no insulin production
• Environmental trigger might be a viral infection
• Major histocompatibility complex at 6p21.3 accounts for 40 % of the familial aggregation.
• HLA-DQ2/DQ8 is present in 30% of type 1 patients
• HLA-DQ6 is notably absent in type I patients: conferring dominant protection [this gene prevents type I diabetes]
• 20 other genes have been implicated
MODY (Maturity-onset diabetes of the young):
• AD an no association with lifestyle
• MODY1-3: single gene type 2 Diabetes Mellitus
• MODY type 2: Glucokinase deficiency (first step in glycolysis): mostly asymptomatic, but 50% of heterozygous women develop
gestational diabetes
• MODY type 1 and 3: hepatocyte nuclear factor 1A and 4A respectively. HNF1A is activator of HNF4A: activation of liver-specific
genes in glucose, cholesterol and fatty acid metabolism
Type II:
• Migration effect is observed in this disease; life style has an effect.
• Transcription factor 7-like 2 (TCF7L2) (consistently replicated in in multiple populations)
• Zinc transporter SLC30A8 (exclusively expressed in insulin-producing beta cells)
• 2 Loci containing genes involved in beta cell development or function
PPAR : Ligand-dependent transcription factor regulating genes involved in lipid and glucose metabolism and adipocyte
differentiation
• Has been implicated in type II diabetes - a common variant (0.8) is associated with diabetes
• Rare pathogenic mutations have been detected in morbidly obese individuals
• Mutation leads to constitutively active PPAR and therefore to increased adipose tissue differentiation
• Thiazolidinediones, Insulin sensitizers are ligands for the PPAR
 88.Genetics of Drug Metabolism
25-30% of prescription drugs are
Codeine
metabolised by CYP2D6,
CYP2C9 or CYPC19
(cytochrome P450s)
Warfarin
Common polymorphisms in the
population affect the efficiency of
each enzyme

Mephenytoin Poor metabolisers of codeine get

no benefit from the drug

Poor metabolisers of warfarin or

(Variants present in all
populations at different mephenytoin are extremely
frequencies - these are for sensitive to the drugs
N. Europeans)
 89. Coronary Artery Disease &
Myocardial Infraction
A common variant on chr. 9p21 (? CDKN2B) predisposes to CAD & MI
- OR ~ 1.3, P < 10-62
- explains ~21% of population risk
Distinct from a 9p21 gene that is associated with T2D

Components of ion channels and ribosomal complexes also implicated

Independent association of common variants at LDLR & PCSK9 (known to affect LDL levels)

Variants also identified that predispose to atrial fibrillation (4q25 - PITX2??) and aortic
aneurysm (OR ~ 1.3)
90. Genetic Screening methods
• FIRST: Performed 11-14 weeks gestation. Maternal serum
free hCG and Pregnancy Associated Protein-A (PAP-A),
Nuchal measurement, Maternal age
Detection Rates:
• 80% fetal Down syndrome
• 90% trisomy 18
• CVS
• Second Trimester Maternal Serum Screening: Measures:
• Alpha-foetoprotein (AFP)
• Human chorionic gonadotropin (hCG)
• Unconjugated estriol (uE3)
• Inhibin-A
Used for detection of:
• Traditionally: ONTDs, Down syndrome, trisomy 18
• Additional: SLO, triploidy, Turner syndrome, IUFD
• Aminocentesis
 FIRST test 10-12 weeks gestation

+ve -ve

CVS for karyotype -ve 2nd trimester

- or free foetal DNA maternal serum
test(?) screening
+ve
+ve
+ve Amniocentesis or
Termination? free foetal DNA (?) -ve
-ve

Latest data shows cell-free DNA testing has

lower false positive rate than FIRST test