Download Molecular Basis of Lung Disease

Molecular Basis of Lung
Disease
Basic Gene Structure
Exons
Polyadenylation
signal
Start of
transcription
Promoter
Initiation
codon
ATG
5’ untranslated
region
Termination
codon
Introns
UAA
UAG
UGA
3’ untranslated
region
HISTORY AND PHYSICAL
FINDINGS
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J.B., a 2-year-old boy, was referred to the pediatric clinic for evaluation of poor growth
During infancy, J.B. had diarrhea and colic that resolved when an elemental formula
was substituted for his standard formula. As table foods were added to his diet, he
developed malodorous stools containing undigested food particles
During his second year, J.B. grew poorly, developed a chronic cough, and had
frequent upper respiratory infections. No one else in the family had poor growth,
feeding disorders, or pulmonary illnesses.
On physical examination, J.B.'s weight and height plotted less than the 3rd percentile
and his head circumference at the 10th percentile. He had a severe diaper rash,
diffuse rhonchi, and mild clubbing of his digits
the pediatrician requested several tests, including a test for sweat chloride
concentration by pilocarpine iontophoresis; the sweat chloride level was 75 mmol/L
(normal, <40 mmol/L; indeterminate, 40 to 60 mmol/L), a level consistent with cystic
fibrosis.
On the basis of this result and the clinical course, the pediatrician diagnosed J.B.'s
condition as cystic fibrosis. J.B. and his parents were referred to the cystic fibrosis
clinic for further counseling, mutation testing, and treatment.
CFTR: CF transmembrane
regulator (positional cloning)
• Since the 1960s, cystic fibrosis (CF) has been one of the
most publicly visible of all human monogenic diseases
• It is the most common fatal autosomal recessive genetic
disorder of children in white populations, with an
incidence of approximately 1 in 2500 white births and a
carrier frequency of about 1 in 25
• The gene cloned by positional cloning in 1989
• Shortly after the CF gene was cloned, physiological
analyses demonstrated that the protein encoded by the
CFTR gene is a regulated chloride channel located in the
apical membrane of the epithelial cells affected by the
disease.
Cystic Fibrosis
Autosomal Recessive
Pedigree illustrating recessive inheritance
Autosomal recessive disease occurs only in individuals with 2 mutant copies, which they
inherited from their normal heterozygous parents (barring the rare new mutation)>
Basic terminology
Genotype: A A
B
(Heterozygous)
(Homozygous)
A
B
A
Gene
Chromosome 6
Maternal copy
DNA
Chromosome 6
Paternal copy
Single gene disorder - determined by the alleles at a single locus
“Salty Baby Syndrome"
Mechanism Underlying Elevated Sodium Chloride Levels in the Sweat of Patients with Cystic Fibrosis
A major pathway for Cl– absorption is through CFTR: The result is that total
sodium chloride flux is markedly decreased, leading to increased salt content
CFTR is expressed in surface epithelium and
serous cells at the base of submucosal glands in a
porcine lung sample
Submucosal glands of a patient with cystic fibrosis
are filled with mucus, and mucopurulent debris
Pseudomonas aeruginosa
A median cross section of a lung from
a patient with CF
The CFTR Gene and Protein
• CFTR gene mapped to
chromosome 7q31
• Spans about 190 kb of DNA
• 27 exons, is predicted to
encode a large integral
.
membrane protein of about
170 kD
• Has five domains: two
membrane-spanning
domains, each with six
transmembrane sequences;
two nucleotide (ATP)-binding
domains; and a regulatory
domain with multiple
phosphorylation sites
no chloride-channel function
lead to misfolded CFTR protein that is prematurely degraded
Recommended mutations to be screen in
CFTR gene
TMD1
TMD2
NBD1
NBD1
R domain
0
1
R117H
R117C
50
23
100
4 5 7 9 10
6a 8
6b
150
11 13 14b
12 14a 15
19
16 18
17a
17b
A455E
R33W
R347C
R347P
F508
I507
G542X
R553X
G493X
G551D
G551S
200kb
R1066H
R1066C
R1066L
20 21
W1282X
S1255X
3849+10kbC>T
22 24
23
N1303K
W1316X
A total of 33 mutations had been screen
including the recommended 25 in our patients
using OLA .
G85E
394delTT
621+1G>T
1898+1G>A
3849+10kbC>T
711+1G>T
2183AA>G
I148T
621+1G>T
R117H
A455E
1717-1G>A
3905insT
W1282X
2789+5G>A
2184delA
1
23
R347H
R347P
R334W
1078delT
4 5
9 10
7
8
V520F
I507del
Δ F508
11 13 14b 16
12
15
S549R
S549N
G542X
G551D
R553X
R560T
18
19
20 21
22 24
23
N1303K
3876delA
3120+1G>A
3659delC
R1162X
OLA Principle
OLA with
mutant DNA
OLA with
normal DNA
mutant
normal
A
normal
G
Fluorescent dye
Fluorescent dye
G
mutant
PO4 common
A
C
PO4 common
T
mutant DNA target
normal DNA target
ligation
ligation
G
A
Normal OLA product
mutant-OLA product
Is slightly longer than
normal OLA product due to
longer mobility modifier
Target Specific Probe
With Mobility Modifier
Result on 3100:
normal
heterozygous
homozygous mutant
Example on Oligonucletide Ligation Assay
Results
∆F508/∆F508
∆F508/∆F508
∆F508
∆F508/-
∆F508
G85E
∆F508/G85E
Allelic frequencies of mutations in the Jordanian
CF patients
10.3%
3.9%
5.2%
G85E
W1282X
711+1G>T
1
23
4 5
9 10
7
8
Δ F508
24.6%
11 13 14b 16
12
15
18
19
20 21
22 24
23
N1303K
1.3%
CFTR gene sequencing is the choice
77 patients with sweat chloride value more than 60 mmol/L
30 tested –ve for
the 33 mutations
47 tested +ve for
the 33 mutations
Heterozygous
(24)
Homozygous
(18)
Compound
Heterozygous
(5)
SERPINOPATHIES
Serine Proteinase Inhibitors
α1-antitrypsin
Alpha-1Antitrypsin Deficiency
and Conformational Instability
Pathologic polymerization of the variant alpha-1antitrypsin
before its secretion from hepatocyte.
Mechanism of Inhibition of
Proteases by Serpins
neutrophil elastase
The main function of α1-antitrypsin is to
protect the tissues, and especially the elastic tissue of
the lungs, against the enzyme neutrophil elastase
Z allele
• One in 10 people of European descent is a carrier of one of two
mutations in alpha1-antitrypsin that result in a partial deficiency of
the inhibitor
• S mutation (Glu264Val), which in homozygotes results in a 40
percent decrease in plasma alpha1-antitrypsin concentrations. This
by itself poses a negligible threat to health,
• but the S variant becomes important if it is coinherited with the more
severe Z mutation (Glu342Lys). Some 4 percent of Northern
Europeans (3 percent in the United States) carry the Z mutation; in
homozygotes, this results in an 85 percent deficit in plasma alpha1antitrypsin concentrations
• Consequently, the plasma concentrations of alpha1-antitrypsin in
both ZZ homozygotes and SZ compound heterozygotes are
insufficient to ensure lifetime protection of the lungs from proteolytic
damage, especially in smokers.
The effect of smoking on the survival of patients with α-1antitrypsin deficiency
Cancer as a genetic disease
• whether sporadic or familial, cancer is fundamentally due
to mutation in various genes controlling cell growth or cell
death
• once initiated, the cancer evolves by accumulating
additional mutations in other genes
• leads to an ever-worsening cascade of mutations
•Original clone of neoplastic cells can evolve into
numerous sublineages with different but overlapping
mutations
Tumor suppressor gene
Protooncogene
Classifying the genes involved
in cancer
Oncogenes – mutant forms of genes
(proto-
•
oncogenes) that positively regulate cell proliferation
and cell survival -usu dominant, gain-of-fn mutations
Tumor suppressors – genes which function to block
•
tumor development by negatively regulating cellular
growth-usu need loss of both copies
Cellular maintenance genes – responsible for the
detection and repair of genetic damage in cells
•
RET, MET
Ras, Abl
Myc
Receptor tyrosine kinases
-transduce an extracellular
signal inward
-bind a ligand, conformational
change that results in kinase
activity, leading to phosphorlation of cellular proteins
-pt mut’s cause receptors to
be constitutively active
RET mut-multiple endocrine
neoplasia
MET mut-hereditary papillary
renal carcinoma
RAS family of proto-oncogenes
• One of the first activated oncogenes discovered by the
DNA transformation assay
• Encodes a small guanosine triphosphate (GTP) –
binding protein (G-protein)
• 3 members of this family; H-RAS, K-RAS, N-RAS
• Serves as an “on/off” switch to activate or inhibit
downstream molecules when bound to GTP
• The protein’s effect is ended by self-directed cleavage
of GTP
RAS family of proto-oncogenes
• Ras associates with the plasma membrane
• Ras relays signals from the cell surface receptors to
the nucleus, functioning as a switch
– ‘Active’ when GTP is bound
– ‘Inactive when the hydrolyzed GDP is bound
•
RAS oncogene activation by
nucleotide
substitution
Conversion to oncogene usually due to a point
mutation in the gene where:
– the ras protein is able to signal continuously, even in
absence of GTP
– the ras protein is unable to hydrolyze GTP to turn
“off” the signal
• Leads to the continuous activation of multiple
downstream signaling pathways inducing cell
proliferation
• Mutations in the 3 RAS genes are found in 10-15% of
all human cancers
RAS mutation in human
cancers
• H-RAS mutated in 10% of all bladder cancer
• K-RAS mutations in about 50% of
colorectal cancers, 70-90% of pancreatic
cancers and 30% of lung adenocarcinomas
as well as in ovarian, breast skin liver and
kidney
• N-RAS mutations have been detected in 2030% of acute nonlymphocytic leukemias
Codon 12 G-T
The BRAF–mitogen-activated and extracellular-signal regulated kinase kinase (MEK)
–extracellular signal-regulated kinase (ERK) cascade often determines
proliferation and becomes deregulated in certain cancers
EGFR in lung adenocarcinoma in approximately 10% of specimens from
patients in the United States and in 30 to 50% of specimens from patients in
Asia. The mutations occur with increased frequency in women and
nonsmokers
Tumor Suppressors: P53
The two-hit hypothesis
•
Tumorigenesis requires loss of function of both copies
of tumor suppressor
•
At clinical level – dominant inheritance (looks like only
a single mutation is required)
•
At cellular level – two mutations required for tumor to
develop
•
First hit may be inherited or occur somatically
•
Second hit occurs somatically
•
One mechanism – loss of heterozygosity - LOH:
individual is heterozygous in normal tissues at a
specific marker but tumor cells contain only 1 of the 2
alleles
The distribution of missense
mutations in human P53 gene
Microarray
Five-gene signature is closely associated with
relapse-free and overall survival
among patients with NSCLC
• NEJM 356;1 www.nejm.org january 4,
2007
• DUSP6, MMD,STAT1, ERBB3, and LCK
MicroRNA (miRNA) genes
MicroRNA signatures in human cancers
Examples of microRNA profiles in human solid and liquid cancers
Facts about microRNA-expression profiling in human cancers
MiRNA profiling as a prognostic
tool
• The expression of both miR-155 (high levels) and let-7a2 (low levels) has been shown to correlate with poor
survival in 104 United States patients with lung cancer.
• In an independent study of 143 Japanese patients with
lung cancer, reduced let-7 expression was found to
significantly correlate with a shorter survival time after
potentially curative surgery
• Ras overexpression was associated with a reduced
survival time. As let-7 negatively regulates Ras, these
results show the existence of a link between let-7, Ras
expression and the life expectancy of patients with lung
cancer.