Transcript Mutations changes of genetic information

The Pathogenesis of Diseases from
Genetic and Genomic Point of View
Oliver
Rácz and František Ništiar
Institute of Pathological Physiology
Medical School, Šafárik University
2012/2013
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26th june 2000
is neither the beginning nor the end of
the way
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5 years before term (1990 - 2005)
The race is over, victory for Craig Venter.
The genome is mapped* - now what ?
 Not a discovery!
 A very important technological result and
competition is always useful.
all is based on Mendel‘s and Watson‘s & Crick‘s
discoveries in XIXth XXth century
*3*109 letters
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Mendel, Watson, Crick & the medical
genetics of XIXth – XXth century

Mendel‘s laws are valid also today

Watson & Crick provided the material basis of
these laws (central dogma of molecular biology)

Mendel‘s laws in medicine can be applied to
monogenic diseases – long list, relatively rare

What is the genetics of diabetes, hypertension,
coronary heart disease, Alzheimer disease ?
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White flower from red
parents ???
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Quidditch ball for Harry Potter ?
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We discovered
the secret of
life, let’s have a
beer!
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The classics
Mendel G: Versuche über PflanzenHybriden. Verh. Naturfortsch. Verein
Brünn, 4, 1866, 3-47
 Watson.JD, Crick FHC: Molecular
structure of nucleic acids. Nature
171, 1953, 737-738

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Central dogma of molecular
biology (cca. 1965)
Replication
Transcription
Translation
Transformation
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Central dogma of molecular
biology
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Central dogma of molecular biology?
Epigenetics ? Role of RNA?
•Regulation of transcription
•Transcription factors, etc.
•Methylation, acetylation…
•Regulation of RNA editing
•Alternative splicing
•Regulation of RNA transport
•Regulation of translation
•siRNA, tRNA modifications
•Postsynthetic modifications
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Central dogma of molecular biology
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Central dogma of molecular
biology (now) – role of RNAs
Replication
Transcription
Translation
Transformation
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It is a little more complicated
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We and our relatives
Organism
Homo sapiens
Mus musculus
D. Melanogaster
C. Elegans
S. Cerevisiae
E. Coli
HIV virus
A.
Thaliana
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Genome size
3 000 000 000
2 600 000 000
137 000 000
97 000 000
12 100 000
4 600 000
9 700
100
000 000
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chromosomes/
genes
23/30 000
20/30 000
4/13 000
6/19 000
18/6 000
{1}3 200
9
?/25 000
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What is not “genetic”
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The number of human genes is as low as 30 000
 the small worm C. elegans has 20 000 genes
 the mouse has as many genes as we, also with very similar
function
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The mystery is in complexity and networking:
230000 >>>> 220000 (possible on/off states)
And a number of surprises around transcription
and translation (miRNA, tRNA modifications)
It is mapped but do we understand it?
GENETICS = HEREDITY
GENOMICS = EVERYTHING
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GENES AND THE ENVIRONMENT
GENOME
ENVIRONMENT
SEVERE MONOGENIC
DISEASES
NEGATIVE AND
POSITIVE
ENVIRONMENTAL
FACTORS
physical
chemical
biological
nutrition
life style
LESS IMPORTANT MUTATIONS
GENETIC RISK
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Genes and diseases in practical
medicine
XIXth Century: symptom  diagnosis
sugar in urine = diabetes
XXth Century: symptom 
etiopatogenesis  diagnosis
autoimmune destruction of b cells = dm Type 1
XXIst Century: symptom 
genes and environment 
etiopatogenesis  diagnosis
susceptibility + overeating =
subtypes of dm Type 2
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New era of preventive medicine
Better understanding of disease
pathogenesis
 Targeted, individualized prevention
 Clear, unambiguous arguments
 Healthy genes healthy people
 Genes sana in corpore sano

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Mutations
changes of genetic information
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THREE PRINCIPAL POSSIBILITIES
1. changes in genome not compatible with life
2. development and diversity
3. disease or increased risk fo disease*
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THE BASIC DIFFERENCE FOR HEREDITY:
–
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somatic and germ cell mutations
genome, chromosomal and gene mutations
no genes for diseases! – sickle cell, Alzheimer, diabetes...
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Gene mutations and SNPs*
Point mutations in exons
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–
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silent, missense (AA change) and nonsense (stop)
Frameshift mutation in exons (1,2,4,5...)
Small deletion of triplets (3,6...)
Bigger deletions – transition to chromosomal
aberrations
Mutations in regulatory parts, introns, genes for rtRNA
Variability of repeated sequences - markers
Dynamic mutations – triple repeat mutations
*SINGLE NUCLEOTID POLYMORPHISM
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Monogenic diseases with mendelian
inheritance
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Autosomal recessive
– sickle cell disease thalassemia and other
hemoglobinopathies
– cystic fibrosis
– enzymopathies (inborn errors of metabolism)
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Autosomal dominant
– familiar hypercholesterolemia
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X chromosome linked diseases
– hemophilia A, B; daltonism

and von Willebrand disease, factor V Leiden,
hereditary hemochromatosis...
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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 – 13.10.2013
20 % heterozygotes in somegene13ab.ppt
parts of Africa
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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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Occurrence of Hb S in the world
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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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Globin genes
zYzYaa2a1
16p - alfa family
eGgAgYbdb
11p - beta family
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Globin genes - ontogenesis
zYzYaa2a1
eGgAgYbdb
Gower 1 z2e2
Portland z2g2
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Gower 2 a2e2
Fetal a2g2
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Globin genes - adult
zYzYaa2a1
eGgAgYbdb
A = a2b2 (95%)
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b
A = a2b2 (95%)
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GgAgYbdb
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Beta thalassemia
zYzYaa2a1
eGgAgYbd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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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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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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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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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
8
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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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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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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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
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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Disroder of color vision
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