Molecular Basis of Lung Disease

Basic Gene StructureBasic Gene Structure

PromoterPromoter

Initiation Initiation codoncodonATGATG

Start of Start of transcriptiontranscription

Termination Termination codoncodon

UAAUAAUAGUAGUGAUGA

Polyadenylation Polyadenylation signalsignal

5’ untranslated 5’ untranslated regionregion

3’ untranslated 3’ untranslated regionregion

ExonsExons

IntronsIntrons

HISTORY AND PHYSICAL FINDINGS

• 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

Pedigree illustrating recessive inheritance

Autosomal Recessive

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 terminologyBasic terminologyGenotype: Genotype: A AA A(Homozygous)(Homozygous)

AA AA

Genotype: Genotype: A A BB(Heterozygous)(Heterozygous)

AA BB

Single gene disorder - determined by the alleles at a single locusSingle gene disorder - determined by the alleles at a single locus

Chromosome 6 Chromosome 6 Maternal copyMaternal copy

DNADNA

GeneGene

Chromosome 6 Chromosome 6 Paternal copyPaternal copy

“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

A median cross section of a lung from a patient with CF

Pseudomonas aeruginosa

.

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

lead to misfolded CFTR protein that is prematurely degraded

no chloride-channel function

Recommended mutations to be screen in CFTR gene

0 50 100 150 200kb

1 2 4

5 7 1093 11 13 14b 16 18 19 20 21 226a6b

8 12 14a 15 17a17b

2324

TMD1 TMD2NBD1 NBD1

R domain

R117HR117C R33W

R347CR347P

F508I507

G542XR553XG493X

G551DG551S

R1066HR1066CR1066L

W1282XS1255X

N1303KW1316X

A455E

3849+10kbC>T

A total of 33 mutations had been screen including the recommended 25 in our patients

using OLA.

1 2 4

5 7 1093 11 13 14b 16 18 19 20 21 228 12 15 23

24

3905insTW1282X

I148T621+1G>T

R117H

R347HR347PR334W1078delT

A455E

V520FI507delΔ F508

S549RS549NG542XG551DR553XR560T

2183AA>G

3659delCR1162X

N1303K3876delA

621+1G>T

711+1G>T

1717-1G>A 2789+5G>A

3120+1G>A

1898+1G>AG85E394delTT

2184delA

3849+10kbC>T

OLA PrincipleOLA with

mutant DNA

mutant

normal

mutant DNA target

ligation

mutant-OLA productIs slightly longer than normal

OLA product due to longer mobility modifier

G

Acommon

T

A

OLA withnormal DNA

mutant

normal

normal DNA target

ligation

Normal OLA product

A

common

G

C

Result on 3100:

normal homozygous mutantheterozygous

Fluorescent dye Fluorescent dye

PO4GPO4

Target Specific ProbeWith Mobility Modifier

Example on Oligonucletide Ligation Assay Results

∆F508

∆F508

G85E

∆F508/∆F508

∆F508/∆F508

∆F508/-

∆F508/G85E

1 2 4

5 7 1093 11 13 14b 16 18 19 20 21 228 12 15 23

24

Δ F508 N1303K

711+1G>TG85E W1282X

10.3% 5.2%

3.9%

1.3% 24.6%

Allelic frequencies of mutations in the Jordanian CF patients

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

Alpha1-Antitrypsin Deficiency and Conformational Instability

Pathologic polymerization of the variant alpha1-antitrypsin before its secretion from hepatocyte.

Mechanism of Inhibition of Proteases by Serpins

neutrophil elastase

The main function of α1-antitrypsin is toprotect 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 alpha1-antitrypsin 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 α1-antitrypsin deficiency

Cancer as a genetic diseaseCancer as a genetic disease• whether sporadic or familial, cancer is fundamentally due whether sporadic or familial, cancer is fundamentally due to mutation in various genes controlling cell growth or cell to mutation in various genes controlling cell growth or cell deathdeath

• once initiated, the cancer evolves by accumulating once initiated, the cancer evolves by accumulating additional mutations in other genesadditional mutations in other genes

• leads to an ever-worsening cascade of mutationsleads to an ever-worsening cascade of mutations

•Original clone of neoplastic cells can evolve into Original clone of neoplastic cells can evolve into numerous sublineages with different but overlapping numerous sublineages with different but overlapping mutationsmutations

Tumor suppressor geneTumor suppressor gene Protooncogene Protooncogene

Classifying the genes involved Classifying the genes involved in cancerin cancer

•OncogenesOncogenes – – mutant forms of genes (proto- mutant forms of genes (proto-

oncogenes) that positively regulate cell proliferation oncogenes) that positively regulate cell proliferation

and cell survival and cell survival -usu dominant, gain-of-fn mutations-usu dominant, gain-of-fn mutations

•Tumor suppressorsTumor suppressors – genes which function to block – genes which function to block

tumor development by negatively regulating cellular tumor development by negatively regulating cellular

growth-growth-usu need loss of both copiesusu need loss of both copies

•Cellular maintenance genesCellular maintenance genes – responsible for the – responsible for the

detection and repair of genetic damage in cellsdetection and repair of genetic damage in cells

RET, METRET, METReceptor tyrosine kinasesReceptor tyrosine kinases-transduce an extracellular-transduce an extracellular signal inwardsignal inward-bind a ligand, conformational -bind a ligand, conformational change that results in kinase change that results in kinase activity, leading to phosphor-activity, leading to phosphor-lation of cellular proteinslation of cellular proteins-pt mut’s cause receptors to-pt mut’s cause receptors tobe constitutively activebe constitutively activeRET mut-multiple endocrine RET mut-multiple endocrine neoplasianeoplasiaMET mut-hereditary papillary MET mut-hereditary papillary renal carcinomarenal carcinoma

Ras, AblRas, Abl

MycMyc

RAS family of proto-oncogenesRAS family of proto-oncogenes• One of the first activated oncogenes discovered by the One of the first activated oncogenes discovered by the

DNA transformation assayDNA transformation assay

• Encodes a small guanosine triphosphate (GTP) –Encodes a small guanosine triphosphate (GTP) –binding protein (G-protein)binding protein (G-protein)

• 3 members of this family; H-RAS, K-RAS, N-RAS3 members of this family; H-RAS, K-RAS, N-RAS

• Serves as an “on/off” switch to activate or inhibit Serves as an “on/off” switch to activate or inhibit downstream molecules when bound to GTPdownstream molecules when bound to GTP

• The protein’s effect is ended by self-directed cleavage The protein’s effect is ended by self-directed cleavage of GTPof GTP

RAS family of proto-oncogenesRAS family of proto-oncogenes• Ras associates with the plasma membraneRas associates with the plasma membrane

• Ras relays signals from the cell surface receptors to Ras relays signals from the cell surface receptors to the nucleus, functioning as a the nucleus, functioning as a switchswitch

– ‘‘Active’ when GTP is boundActive’ when GTP is bound

– ‘‘Inactive when the hydrolyzed GDP is boundInactive when the hydrolyzed GDP is bound

RAS oncogene activation by RAS oncogene activation by nucleotide substitutionnucleotide substitution

• Conversion to oncogene usually due to a point Conversion to oncogene usually due to a point mutation in the gene where:mutation in the gene where:

– the ras protein is able to signal continuously, even in the ras protein is able to signal continuously, even in absenceabsence of GTP of GTP

– the ras protein is the ras protein is unable to hydrolyze GTPunable to hydrolyze GTP to turn to turn “off” the signal“off” the signal

• Leads to the continuous activation of multiple Leads to the continuous activation of multiple downstream signaling pathways inducing cell downstream signaling pathways inducing cell proliferationproliferation

• Mutations in the 3 RAS genes are found in 10-15% of Mutations in the 3 RAS genes are found in 10-15% of all human cancers all human cancers

RAS mutation in human RAS mutation in human cancerscancers

• 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 20-30% of acute nonlymphocytic leukemias

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

Codon 12 G-T

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: P53Tumor Suppressors: P53

The two-hit hypothesisThe two-hit hypothesis• Tumorigenesis requires loss of function of both copies Tumorigenesis requires loss of function of both copies

of tumor suppressorof tumor suppressor

• At clinical level – dominant inheritance (looks like only At clinical level – dominant inheritance (looks like only a single mutation is required)a single mutation is required)

• At cellular level – two mutations required for tumor to At cellular level – two mutations required for tumor to developdevelop

• First hit may be inherited or occur somaticallyFirst hit may be inherited or occur somatically

• Second hit occurs somaticallySecond hit occurs somatically

• One mechanism – loss of heterozygosity - LOH: One mechanism – loss of heterozygosity - LOH: individual is heterozygous in normal tissues at a individual is heterozygous in normal tissues at a specific marker but tumor cells contain only 1 of the 2 specific marker but tumor cells contain only 1 of the 2 allelesalleles

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-7a-2 (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.