Download Mutations changes of genetic information

The Pathogenesis of Diseases from
Genetic and Genomic Point of View
II.
Oliver Rácz
Institute of Pathological Physiology
Medical School, Šafárik University
2009 – 2016
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Sickle cell disease
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Clinical description  1910, Hb abnormality, 1940
Pauling / Ingram - 1 AA change in b chain
Point mutation – Glu  Val on 6th place (GAG/GTG)
Decreased solubility of Hb in low pO2
Rigid, deformed red cells in venous blood
Thrombosis, decreased life span of Er, hemolysis,
icterus, anemia - HYPOXIA
Epidemiology: 8 % of black people in USA are heterozygotes;
1:400 homozygotes
5 – 26.9.2016
20 % heterozygotes in some gene16ab
parts of Africa
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The molecular structure of human Hb
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Normal and sickled red cells
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The pathogenesis of sickle cell disease
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Occurrence of Hb S in the world
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Treatment 2016
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Symptomatic???
Antisickling agents (toxic)
Almost healthy sickle child???
– TWO MUTATIONS: Hb S AND PERSITENCE OF
FETAL Hb!
– Cca 20% is enough to prevent sickling
– Artificial induction of F Hb?
CRISPR repair of zygote?
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And the other haemoglobinopathies ?
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Theoretical number – astronomical
Known  500, very rare
Hb C = same point as Hb S but lysine, mild
haemolysis. HbSC heterozygotes
Different types: labile, low and high oxygen affinity,
methemoglobin formation, etc.
Why is sickle cell disease relative common?
Plasmodia malariae do not like Hb S!
AA dies on malaria, SS on sickle cell disease
AS have relative advantage for survival
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Globin genes
z
Yz
Ya
a2
a1
16p - alfa family
e
Gg
Yb
Ag
d
b
11p - beta family
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Globin genes - ontogenesis
z
e
Gower 1 z2e2
Portland z2g2
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Gower 2 a2e2
Yz
Gg
Ya
Yb
Ag
a2
d
a1
b
Fetal a2g2
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Globin genes - adult
z
e
Yz
Gg
A = a2b2 (95%)
Ya
Yb
Ag
a2
d
a1
b
3 exons and 2 introns
A2 = a2d2 (1%)
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Globin genes – regulation!
LCR = locus control region with
promoter and 2 enhancers before the
cluster
LCR deletion – no gama/delta/beta chain
synthesis
e
Gg
Ag
Yb
d
A = a2b2 (95%)
b
3 exons and 2 introns
A2 = a2d2 (1%)
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Thalassemias
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Deletion of smaller or bigger parts of a or b gene
region
(or mutation in regulatory parts, nonsense
mutation and intron splicing mutations)
b deletions – back to embryonal Hb F if possible
a deletions – 2*2 = 4 genes!
aaaa
aaa
aa
a
no a
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norm, healthy
1 deletion, clinically not manifest
2 deletions, clinically mild/not manifest
thalassemia Hb Bart = g4
hydrops fetalis
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Alpha thalassemias
z
Yz
Ya
a2
a1
2-krát
e
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Gg
Yb
Ag
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d
b
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Beta thalassemia
z
e
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Yz
Gg
Ya
Yb
Ag
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a2
d
a1
b
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Cystic fibrosis
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Woe to that child which when kissed on the
forehead tastes salty. He is bewitched and soon
must die
Anders, 1938 – cystic fibrosis of pancreas
Farber, 1945 - mukoviscidosis
SR 3 centres – BA, BB, KE (cca 60)
1/26 heterozygotes, 1/2736, (1/676 marriages)
Increased NaCl is sweat, thick secrets of glands in
bronchi, pancreas, GIT - organ failure, death
Disorder of reverse chloride and water transport
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Clinical manifestation
(Spišák, Feketeová)
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TYPICAL SPTs
– Progressive bronchopulmonal
disease
– Nasal polyps
– Pancreas insufficiency
– Meconium ileus
– Male infertility
– Malnutrition
– Growth retardation
– Rectal prolaps
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ATYPICAL SPTs
– Icterus
– Distal gut obstruction
– Liver and bile tract
malfunction
– Pankreatitis
– Chronic rhinosinusitis
– Diabetes mellitus
Spišák B, Feketeová A: Cystická fibróza
Pediatria 1, 2006,, 194-198
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Cystic fibrosis competition
Willamson et al., London 1987 - miss
Lap-Chee Tsui a spol, Toronto, 1989 - hit
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Very different situation compared to Hb S
– The faulty protein was not known
– The localization of the gene was unkown
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Genetic linkage with an enzyme polymorphism chromosome 7 (classical genetics)
Further markers, narrowing down of the region
4 clones, 1 complementary with cDNA* of a
protein from sweat gland
Localization and sequenation of the gene
*cDNA = mirror of mRNA for a synthesized protein
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Cystic fibrosis
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Different from haemoglobinopathies:
– No known protein involved
– Unknown site for mutation
Genetic linkage with an enzyme polymorphism
located on ch. 7
Further markers in the region
4 clones, 1 is complementary to a sequence from
sweat gland
the gene is found and sequenced
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Cystic fibrosis
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CFTR (cystic fibrosis conductance regulator gene)
1989, chromosome 7 - a big gene with 24 exons
Codes a big transmembrane protein - Cl channel
More than 1000 mutations found in the gene
BUT
60 % patients have a triplet deletion - omission a
Phe on 508th place of protein
additional 15 % 8 other mutations (also in introns)
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Cystic fibrosis
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Norm: ATC TTT = Ileu Phe
Deletion: ATC TTT
ATT = Ileu, deleted CTT but lack of TTT
Prenatal diagnostics – direct?
Indirect – something is wrong with the
gene
NaCl in sweat > 60 mmol/l
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The structure of chloride transporter coded by
CFTR gene
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The structure of chloride transporter coded by
CFTR gene
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CFTR protein
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2 transmembrane domains (Cl transport)
2 nucleotide binding domains (for ATP)
Regulation domain R
REGULATION ALSO OF OTHER CHANNELS !!!
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Different functions in sweat and other glands !!!
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CFTR is a chloride channel
BUT
Its functions are tissue specific
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Sweat glands – salty sweat
Cooperation with ENaC, NaCl in sweat low
No CFTR, salty sweat
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Other exocrine glands
thick secrets
Cooperation with ENaC, influences of water
transport
(and ofj bicarbonate)
No CFTR, thick acid secrets
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How many cystic fibroses we have?
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Typical monogenous disease (?)
The number of known mutations in CFTR gene >
1300
66 % of patients have a deletion of a triplet in
10th exon = deletion of Phe 508, the protein is
degraded in the endoplasmic reticulum
20 other mutations (also in introns) – other 15 %
patients
6 different classes of mutations – different clinical
symptoms of also without
Mixed heterozygotes !!!
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Cystic fibrosis
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Gene therapy – insertion of the gene with viral
vectors into airway epithelial cells – not a real
success
Treatment of infections, dilution of mucus,
improvement of the digestion
Physiotherapy
Psychosocial care
Lung transplantation
SURVIVAL 1975 10 – 15 years, today > 40
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Mixed heterozygotes
508/508
508/other
Other/other
No
151
117
25
%
52
40
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Panreas
99%
insuficiency
Pancreas ok 1%
72%
36%
28%
64%
Age at dg
4,4 y
8,4 y
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1,8 y
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Cystic fibrosis and personalized
medicine?
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Ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)1,4-dihydro-4-oxoquinoline-3-carboxamide
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The G551D-CFTR mutation results in defective channel
opening, present in 4% of patients with CF, affecting
approximately 1,200 people in the US. In vitro studies
utilizing CF human bronchial epithelial cells positive for
G551D have shown that ivacaftor increases stimulated
chloride secretion and also reduces excessive sodium and
fluid absorption
 PRICE
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??? OTHER CHILDREN ???
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Hemophilia A
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Talmud
Queen Victoria and her descendants ?
Family of the last Russian Czar Nicolaus
(Alexandra - 4 daughters and one affected son)
Absolute deficiency of factor VIII
1/10000 boys, one third are new mutations in their
ancestors (during meiosis)
High number of mutations, the most common
form is an inversion with 0 activity of factor
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Hemophilia A
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Epidemiology and the Queen
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172 000 worldwide
» 60% severe, classic (gene inversion)
» 15% moderate (1-5 % activity)
» 25% mild (6-30%)
Moderate and mild, more “B” and other
Queen Victoria 1819 – 1901, 9 children
Carriers Alice and Beatrice (2/5)
Affected Leopold, 1853 – 1884 (1/4)
Edward VII healthy, etc… George
Alice, Alexandra and Alexei (1904 – 1918)
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The Queen and her Russian
grand-grandson
Queen Victoria 1819 – 1901, 9 children
Carriers Alice and Beatrice (2/5)
Alices daughter, Alexandra/Nicholas
4 daughters Olga, Tatiana, Maria,
Anastasia and affected Alexei (1904 –
1918)
1918 Tobolsk, execution
THEY ALL HAD MUTATION OF
FACTOR IX = HEMOPHILIA B
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History of treatment
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1840 – first attempt with transfusion
1923 – plasma replacement therapy
1960 – 70 cryoprecipitate, 40 000 HIV INFECTIONS
1989 – Genetic engineering, pure VIII and IX
2014 – Extended life of factors
In progress – Gene therapy
Not only queens but also dogs have haemophilia
Treatment of immune reaction and joint damage
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Structure of factor VIII and IX genes and proteins
(with vWf)
F VIII
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F IX
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Other coagulopathies
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Haemophilia B - similar to A, not so serious
Haemophilia C - AR heredity
All other factors - very rare
Von Willebrand disease - AD; mild or
asymptomatic, heterogeneous
vW factor is a big protein with multiple function stabilizes factor VIII
Bleeding when associated with other
circumstances (acetylsalicylic acid)
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An „upside down“ coagulopathy
Hereditary thrombophilia
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Point mutation in factor V (Leiden)
The protein is resistant to thrombolytic
inactivation.
Part of common european heritage (2-7 %)
Elevated risk of venous thrombosis:
VV = 1; vV = 7; vv = 80;
Manifestation in association with other
circumstances
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Disorder of color vision – daltonism
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Francis Dalton, Manchester, fyzik (1776-1844)
Did not understand why he perceived the colors
differently as other people and let his eyes conserved
in formaline
4 photoreceptors (G-proteins, Guiness recored in
sensitivity), vitamin A
Genes for red and green opsins are on the X, 98 %
homolog, polymorph
8 % white men (no selection pressure)
Gene analysis from Dalton’s retina – 1992
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Disorder of color vision
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Dominant heredity ?
Simple?
Example: polydactylia
Gene mathematician (5)
Stupid in math (6)
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Polydactylia in cats of sailors and of Ernest
Hemingway
Sonic hedgehog – an important gene regulating
development of different body parts – not only
extremities and fingers.
(discovered in drosophila and working also in
fish)
Coding a small signal molecule
Point mutation in the regulatory region of the
gene makes a small mistake in us, in cats,
chicken, mice, horses...
Homozygotes – serious disorders of body
development
Gene knockaut: no development of limbs at all
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