Minist erul Sănătății al Republicii Mld ova [600156]

Minist erul Sănătății al Republicii Mld ova
IP. Univ ersitatea de Stat de Medicină și F armacie "Nic olae Testemițanu"

Medicin e faculty Nr.2
Department of molecular biology and hum an genetics

TEZA DE DIPL OMĂ
„ROLUL F ACTORILOR GENETICI ÎN D EZVOLTAREA
CARDIOMIOPATIEI HIP ERTR OFICE”

Competitor: Mele veettil m ohammed
raneesh
studenta anului VI, Group M1042
Scientific advisor: Cemortan Igor
doctor în bi ologie, conferențiar univ ersitar
Consult ant: Gr ejdieru Alexandra
conferențiar univ ersitar,d.m.

Chișinău 2016

CONTENTS
List of Abbreviations ………………………….. ………………………….. ………………………….. ………………………….. ……….. 3
INTR ODUCTI ON ………………………….. ………………………….. ………………………….. ………………………….. ……………… 4
SCOPUL LUCRĂRII ………………………….. ………………………….. ………………………….. ………………………….. …………… 6
OBJECTIV ES OF TH E STUDY ………………………….. ………………………….. ………………………….. ………………………….. 6
Însemnăt atea științifică: ………………………….. ………………………….. ………………………….. ………………………….. ….. 6
Însemnăt atea practică: ………………………….. ………………………….. ………………………….. ………………………….. ……. 7
Chapter I. Lit erature review ………………………….. ………………………….. ………………………….. ……………………….. 7
1.1. Basics of CFTR g ene ………………………….. ………………………….. ………………………….. ………………………. 7
1.2. Basics of CFTR g ene and pr otein ………………………….. ………………………….. ………………………….. …….. 9
1.3. PATHOGENESIS ………………………….. ………………………….. ………………………….. ………………………….. . 10
1.4. CFTR activity and ph enotype: Total CFTR activity ………………………….. ………………………….. ………… 20
CF-causing mut ations on both alleles ………………………….. ………………………….. ………………………….. ………. 20
Spectrum of phenotypes associated with t otal CFTR activity ………………………….. ………………………….. …… 21
Spectrum of phenotypes associated with CFTR activity of both alleles ………………………….. …………………. 22
1.5. Clinic al presentation ………………………….. ………………………….. ………………………….. ……………………. 22
1.6. Methods of diagnosis ………………………….. ………………………….. ………………………….. ………………….. 25
Conclusi on of chapter I ………………………….. ………………………….. ………………………….. ………………………….. 27
CHAPTER II. R esults and Discussi ons ………………………….. ………………………….. ………………………….. ……………. 28
3.1. C omparative study b etween population of Moldova and Indi a ………………………….. ………………….. 28
3.2. Recommendation for diagnosing and scr eening ………………………….. ………………………….. …………. 28
3.3. Management of Cystic Fibr osis ………………………….. ………………………….. ………………………….. …….. 30
Conclusi on of chapter II ………………………….. ………………………….. ………………………….. …………………………. 33
GENERAL CONCLUSI ON ………………………….. ………………………….. ………………………….. ………………………. 34
BIBLI OGRAFIE ………………………….. ………………………….. ………………………….. ………………………….. ………………. 35
DECLARAȚIE ………………………….. ………………………….. ………………………….. ………………………….. …………………. 36

List of Abbreviations

 CF :Cystic Fibr osis
 CFTR :Cystic Fibr osis Tr ansmembrane Conduct or Regulator
 Bp :Base pair
 Pter :promoter
 mRN A :Messenger RN A
 ER :Endoplasmic R eticulum
 ABC :ATP Binding C assette
 cAMp :Cyclic AMP
 CBAVD :C ongenital Bilateral Absence of the Vas Deference
 GI :G astro Intestinal

INTR ODUCTION
Definiti on
Cystic fibr osis is an autosomal recessive disease caused by mutations in th e cystic
fibrosis tr ansmembraneराconduct ance regulator(CFTR) g ene.
CFTR g ene encodes the protein called transmembrane conduct ance regulator which
functi ons as a chloride channelराfor the transporting of chloride ions across epithelial
cellsराon muc osal surf ace.CFTR pr otein also has inhibit ory effect on sodium
channels. CF is th e most common life threatening g enetic condition in th e Caucasian
population.
Epidemiology
Cystic fibr osis is th e most common lethal hereditary dis ease in the white population.
In the United States, approximately 30,000 individu als have CF; m ost are diagnosed
by six m onths of age. In Canada, there are approximately 4,000 p eople with CF
Approximately 1 in 25 p eople of European descent, and one in 30 of Caucasian
Americans is a carrier of a cystic fibr osis mut ation. Although CF is l ess common in
these groups, approximately 1 in 46 Hisp anics, 1 in 65 Africans and 1 in
90 Asians carry at least one abnormal CFTR g ene. (1, 2)
In the last tw o decades, CF h as been incr easingly di agnosed in L atin America, the
Middl e East, and populations derived from the Indian subc ontinent that have
emigr ated to Western Europe, thus implying th e presence of CF in signific ant
numb ers among th e citizens of Indi a and Pakistan wh o have remained in th eir
homelands.
Europe
The incid ence of CF is w ell documented in Europe. On average 1 in 2000 -3000 n ew-
born are affected with CF. Even wh ere populations appear relatively homogeneous,

there may be marked local and regional variations. In Fr ance, for example, there is a
very high incid ence of CF in N orthwest Britt any and a lower incidence in the South.
CFTR g ene mutations have been well characterized in m ost European populations. In
several Western European countri es, mut ations are detected in m ore than 95% of the
CFTR g enes derived from CF p atients. Th e F508d el CFTR mut ation is th e most
common mut ation causing CF. F508d el. Frequencies vary from a maximum of 100%
in the isolated Faroe Islands of Denmark, to a minimum of about 20% in Turk ey. In
central, northern, w estern, and north-eastern Europe, F508d el has a frequency of
about 70%. Apart from F508d el, 5 to 10 relatively frequent mut ations contribut e to
10%-15% of all CF -causing CFTR mut ations, such as the G542X, N1303K, and
G551D mut ations. Th e G542X and N1303K mut ations are most common in
Mediterranean countri es. Ethnic sp ecific mut ations are observed in s ome populations
such as the Nordic mut ation 394d elTT, th e 3905insT mut ation in Switz erland, th e
R1162X mut ation in N ortheast Italy, and th e Eastern Sl avic CFTRd ele2,3(21kb)
mutation. Th e remaining mutations are heterogeneous, priv ate, or limit ed to a small
numb er of individu als.(3)
Asia
CF is n ormally rare in Asians and th ere are few reports of CF affected people of
Asian origin. Th e exact incid ence is not known but the predicted incid ence for Asians
in the United Kingd om (m ainly Indi an/Pakistani) is 1 in 10,000 and 1 in 40,000 in th e
USA. In Indi a, the CF incid ence is estimated to be 1 in 40,000 t o 100,000 live births.
In Japan, the estimated incid ence is 1 in 100,000 t o 350,000 liv e births.CF d efinitely
exists in th e Indian subc ontinent but is pr obably less common than in Europe. The
frequency of F508d el reaches about 60% in P akistani CF p atients, but is much l ower
in Indi an (about 20%) and Japanese patients (about 10%).Th ere are other rarer CFTR
mutations in th e Indian population, but n o second fr equent of mut ation could b e
detected, possibly b ecause of the highly h eterogeneous nature of the population.
Although a limit ed numb er of patients h ave been studi ed, no singl e mutation has a

frequency high er than 15%. In studi es of Pakistani patients, s ome rarer mut ations
appear to be relatively frequent, however they are mostly f ound in h omozygous state
because of consanguinity. In J apan, no mutation frequent for clinic ally di agnosed CF
has been found. (4,5,6,7)
India
Since India is the second populous country in th e world and du e to lack of studi es and
non availability of investigations at most centers in Indi a, the precise incid ence of
cystic fibr osis in India is unkn own.As per recent studi es done in vellore medical
college and W orld health organisation, prevalence of cystic fibr osis in Indi a is 1 in
40000 -100000 births.
Indian continent du e to the medical community's l ack of knowledge of the disease,
poor access to medical facilities and health care for CF p atients, c onfounding
diagnosis, a high inf ant mortality r ate, and low life expectancy in g eneral. Respiratory
and gastro-intestinal problems associated with m alnutriti on are very common in
developing c ountri es, and th e diagnosis of CF c an therefore be missed due to a low
index of suspici on.
SCOPE OF WORK
To analyse the genetic mt erial of CF in diff erent population.
OBJECTIV ES OF TH E STUDY
1. To study th e genetic component & mut ation of cystic fibr osis in diff erent races.
2. To pathogenesis g enes that causes cystic fibr osis.
3. To know the distributi on of cystic fibr osis patients.

Însemnăt atea științifică:
A fost realizată o clasificare a factorilor genetici r esponsabili de stabilirea și
evoluția CMH.

Însemnăt atea practică:
1. Au fost obținut e abilități d e manipul are a acizilor nucl eici în l aborator.
2. Au fost analizați arbori genealogici ai familiil or cu CMH .
3. S-a confirm at asocierea polimorfism elor ADN ale genelor ACE și NOS cu
HTA.

Chapter I. LITERATUR E REVIEW
1.1. Basics of CFTR g ene
Location – q31.2 l ocus of chromosome 7
Fig 1 Genomic L ocation for CFTR G ene

Start: 117,465,784 bp fr om pter

End: 117,715,971 bp fr om pter
Size: 250,188 b ases
Orientation: Plus str and or sense strand or coding str and
Cellular Processing : 27 exons encode 1480 amino acid CFTR glyc oprotein with
a molecular weight of 170000. C onstructi on and pl acement of the CFTR pr otein
in the cell membrane occurs in distinct ph ases. Located on the long (q) arm of
chromosome 7 at position 31.2, th e CFTR gene is compris ed of 27 exons th at encode
its genetic sequence. An exon is a portion of a DNA that contains th e code for a
protein structur e. The CFTR g ene is transcrib ed into a singl e strand of RN A within
the cell nucl eus; regions th at are not needed to make the protein are splic ed out,
producing th e final messenger RN A (mRN A).
The mRN A is translated into protein by rib osomes after moving fr om the nucleus to
the endoplasmic r eticulum, or ER. A numb er of proteins called chaperones facilitate
folding of the new CFTR pr otein and its tr ansfer through th e ER. CFTR is th en
furth er processed in th e Golgi apparatus wh ere sugars are added, and th en sent to the
apical surf ace of the cells.
Function: This g ene encodes a member of the ATP-binding c assette (ABC)
transporter sup erfamily. ABC pr oteins tr ansport various m olecules across extra- and
intra-cellular membranes. ABC g enes are divid ed into seven distinct subf amilies
(ABC1, MDR/T AP, MRP, ALD, OABP, GCN20, Whit e). This pr otein is a member
of the MRP subf amily th at is inv olved in multi -drug r esistance.
involved in th e transport of chloride ions. M ay regulate bicarbonate secretion and
salvage in epithelial cells by r egulating th e SLC4 A7 transporter. Can inhibit th e
chloride channel activity of ANO1. Plays a role in the chloride and bic arbonate
homeostasis during sp erm epididym al maturation and capacitation.(8)

1.2. Basics of CFTR g ene and pr otein

Fig 2 -Schematic diagram of CFTR showing its domain organization
CFTR c onsists of two homologous halves, each containing a hexa-helical
membrane-spanning d omain (MSD1 and MSD2) and a nucleotide-binding d omain
(NBD1 and NBD2). Th e two halves are connected by th e R domain.Th e NBDs
contain conserved ATP-binding s equences: Walker A and B m otifs, cl assifying
CFTR as a member of the ATP-binding c assette (ABC) tr ansporter family. Structur al,
biochemical, and functi onal evidence sugg est that the two NBD d omains int eract and
the ATP-binding sit e of one NBD is c omplemented by th e ABC sign ature motif of
the other. Whil e NBD1 f olds largely cotranslationally, th e native fold of NBD2 as
well as CFTR is attained posttranslationally. Assembly of MSD1, NBD1, R d omain,
and MSD2 is n ecessary and suffici ent to form th e minim al folding unit of CFTR
.These and other observations supp ort the cooperative domain folding m odel and
ensure the dynamic c onformational coupling b etween the cytosolic NBDs and th e
pore-forming MSDs in th e native molecule and pr ovide a structur al explanation for
the cooperative domain unf olding, c aused by cystic fibr osis (CF) mut ations.

Location:Outer membrane of cells in th e exocrine secretions in th e body which
includ es sweat glands,lungs,p ancreas,gastrointestinal tracts,sinus es and reproductiv e
systems.
Physi ological effects of CFTR activity
 Sinus es – CFTR pr otein help to regulate salt and w ater balance,which
contribut es to normal mucus hydr ation and cl earance from sin onasal passages.
 Pancreas – CFTR pr otein regulates the transport of chloride and bic arbonate to
maintain a normal volume and pH of pancreatic secretion necessary for
digestion of nutri ents.
 Gastrointestinal tract – CFTR -regulated balance of electrolytes and fluid
allows for normal hydr ation and appropriate intestinal pH as well as normal
composition of intestinal secretion.

 Lungs – CFTR proteins h elps to regulate salt and w ater,which c ontribut es to
normal hydr ation of airways and muc ociliary clearance.

 Reproductiv e system –CFTR pr otein helps regulation of chloride and
bicarbonate which h elps in th e development of vasdeferens in m ales and also it
helps to hydrate cervical mucus in f emales.

 Sweat gland – CFTR pr otein helps chl oride to pass thr ough th e channel
following s odium r eabsorption,to maintain normal chloride levels.
1.3. PATHOGENESIS

Molecular level pathogenesis
Mutations in th e CFTR gene result in abnormalities of cAMP-regulated chl oride
transport across epithelial cells on muc osal surf aces. Tw enty-five years ago, a variant

(p.Ph e508d el; also known as F508d el in legacy nomenclature) in th e cystic fibr osis
transmembrane conduct ance regulator (CFTR) gene was found t o be the most
common cause of cystic fibr osis.

Fig 3 -Molecular consequences of variants in CFTR
Presently, six m ajor classes of mut ations are distinguish ed based on their cellular
pathophysi ology.
I. Complete absence of CFTR pr otein synth esis.
II. Defective protein m aturation and early d egradation (caused by th e most
common mut ation, ΔF508).
III. Disordered regulation (diminish ed ATP binding and hydr olysis).
IV. Defective chloride conduct ance or channel gating.
V. Diminish ed transcripti on du e to promoter or splicing abnormality.
VI. Accelerated channel turn over from th e cell surf ace.

Well over one thousand mut ations have been describ ed that affect the CFTR g ene in
different ways. Th e most common CFTR mut ation is a deletion of just thr ee DNA
nucleotides, which l ead to the deletion of an amino acid(ph enylalanine) at position 508
of the protein sequence. this is d enoted as ΔF508 and is f ound in around 90% of CF
patients.(9,10,11,12)

Fig 4 -Traditional classification of CFTR mutation defects

Gene Mutations

1) ΔF508
The F508d el mut ation is th e most common CFTR mut ation in th e world. In a
numb er of countri es, the prevalence of the homozygous F508d el genotype
among patients with CF is >40% .F508d el has a severe defect in CFTR
processing and trafficking with d egradation of imm ature CFTR pr oteins in th e
endoplasmic r eticulum, typic al of a Class II mut ation. Few to no CFTR
channels are present at the apical cell surf ace . F508d el has also been shown to
be a Class III mut ation, resulting in CFTR pr oteins with r educed channel-open
probability ( or gating) .F508d el also manifests ch aracteristics of a Class VI
mutation so that the few CFTR pr oteins th at reach th e cell surf ace have
decreased surf ace stability.

2) G542X
G542X is th e second m ost common CFTR mut ation in th e world.In th e CFTR2
global database, ~5% of patients with CF h ave at least 1 c opy of the G542X
mutation. 86%5%4%3%2%Major Gene Mutations
ΔF508 G542X G551D A N1303K W1282X

G542X is a nonsense mutation, which pr oduces a premature stop codon. Th e
cell cannot synth esize a full-length CFTR protein, a Class I mut ation. As a
result, f ew to no CFTR pr oteins are present at the apical cell surf ace.

3) G551D
G551D is th e third m ost common CFTR mut ation in th e world.In th e CFTR2
global database, ~4% of patients with CF h ave at least 1 c opy of the G551D
mutation.G551D is a missense mutation that results in CFTR pr oteins with a
severe reducti on in ch annel-open probability ( or gating), a Class III mut ation.

4) N1303K
The N1303K mut ation is th e fourth m ost common CFTR mut ation in th e
world.In th e CFTR2 gl obal database, ~3% of patients with CF h ave at least 1
copy of the N1303K mut ation.N1303K is a missense mutation resulting in a
severe defect in CFTR pr ocessing and trafficking, typic al of a Class II
mutation.Du e to degradation of imm ature CFTR pr oteins in th e endoplasmic
reticulum, CFTR pr oteins fail to reach th e apical cell surf ace.

5) W1282X
W1282X is th e fifth m ost common CFTR mut ation in th e world.In th e CFTR2
global database, ~2% of patients with CF h ave at least 1 c opy of the W1282X
mutation. W1282X is a nonsense mutation, which pr oduces a premature stop
codon. Th e cell cannot synth esize a full-length CFTR pr otein, a Class I
mutation. As a result, f ew to no CFTR pr oteins are present at the apical cell
surface.

6) 621+1G –›T
Country r egistri es listing th e 621+1G –›T mut ation report a 0.2% t o 6%
prevalence among patients with CF.In th e CFTR2 gl obal database, ~2% of

patients with CF h ave at least 1 c opy of the 621+1G –›T mut ation. 621+1G –›T
is a splic e mutation, which pr oduces a premature stop codon.The cell cannot
synth esize a full-length CFTR pr otein, a Class I mut ation. As a result, f ew to
no CFTR pr oteins are present at the apical cell surf ace.

7) R117H
R117H is th e most common residual functi on CFTR mut ation in th e world. In
the CFTR2 gl obal database, ~2% of patients with CF h ave at least 1 c opy of
the R117H mut ation.R117H is a missense mutation. R117H -protein ch annels
reach th e apical cell surf ace, but th eir functi on is d ecreased.R117H pr oduces a
CFTR pr otein with r educed channel-open probability ( or gating), ch aracteristic
of a Class III mut ation.R117H also results in d ecreased chl oride conduct ance,
typic al of a Class IV mut ation.R117H -5T: C auses the greatest reducti on of
functi onal CFTR at the apical cell surf ace. Typic ally, 5T in cis with R117H
(i.e., on the same allele) is associated with a CF ph enotype. R117H -7T: L ess
likely than R117H -5T to result in CF, h owever, individu als can develop CF.
Patients wh o have 7T in cis with R117H m ay still n eed to be monitored for the
development of CF. R117H -9T: Highly unlik ely to result in CF.

8) R553X
Country r egistri es listing th e R553X mut ation report 0.2% t o 2% pr evalence
among patients with CF. R553X is a nonsense mutation, which pr oduces a
premature stop codon. Th e cell cannot synth esize a full-length CFTR pr otein, a
Class I mut ation.As a result, f ew to no CFTR pr oteins are present at the apical
cell surf ace.

9) 1717 -1G–›A
Country r egistri es listing th e 1717 -1G–›A mutation report 0.1% t o 3%
prevalence among patients with CF. In th e CFTR2 gl obal database, ~2% of

patients with CF h ave at least 1 c opy of the 1717 -1G–›A mutation.1717 -1G–›A
is a splic e mutation, which pr oduces a premature stop codon.The cell cannot
synth esize a full-length CFTR pr otein, a Class I mut ation.As a result, f ew to no
CFTR pr oteins are present at the apical cell surf ace.

10) 2789+5G –›A
Country r egistri es listing th e 2789+5G –›A mutation report 0.2% t o 2%
prevalence among patients with CF. In th e CFTR2 gl obal database, ~1% of
patients with CF h ave at least 1 c opy of the 2789+5G –›A mutation.2789+5G –
›A is associated with alternative splicing and reduced synth esis of CFTR
proteins, typic al of a Class V mut ation. This r esults in s ome functi onal CFTR
proteins at the apical cell surf ace.

11) 3849+10kbC –›T
Country r egistri es listing th e 3849+10kbC –›T mut ation report 0.2% t o 2%
prevalence among patients with CF. In th e CFTR2 gl obal database, ~1% of
patients with CF h ave at least 1 c opy of the 3849+10kbC –›T mut ation.
3849+10kbC –›T is associated with alternative splicing and reduced synth esis
of CFTR pr oteins, typic al of a Class V mut ation.This r esults in s ome functi onal
CFTR pr oteins at the apical cell surf ace.

12) R1162X
Country r egistri es listing th e R1162X mut ation report ≤1% pr evalence among
patients with CF. R1162X is a nonsense mutation, which pr oduces a premature
stop codon. Th e cell cannot synth esize a full-length CFTR pr otein, a Class I
mutation.As a result, f ew to no CFTR pr oteins are present at the apical cell
surface.

13) I507d el

Country r egistri es listing th e I507d el mut ation report ≤1% pr evalence among
patients with CF. I507d el has a severe defect in CFTR pr ocessing and
trafficking with d egradation of imm ature CFTR pr oteins in th e endoplasmic
reticulum, typic al of a Class II mut ation.Few to no CFTR ch annels are present
at the apical cell surf ace.

14) G85E
Country r egistri es listing th e G85E mutation report ≤1% pr evalence among
patients with CF G85 E is a missense mutation resulting in a severe defect in
CFTR pr ocessing and trafficking, typic al of a Class II mut ation.Du e to
degradation of imm ature CFTR pr oteins in th e endoplasmic r eticulum, th ey fail
to reach th e apical cell surf ace.

15) 2183 AA––›G
Country r egistri es listing th e 2183 AA––›G mut ation report ≤1% pr evalence
among patients with CF. 2183 AA––›G is a frameshift mut ation, which
produces a premature stop codon.The cell cannot synth esize a full-length
CFTR pr otein, a Class I mut ation. As a result, f ew to no CFTR pr oteins are
present at the apical cell surf ace.(13,14,15)

Fig 5 -CFTR mut ations

For example the above image shows th e mutation in th eir one of 27 exons and
clinic al expression.
CFTR mutations have poor penetrance. This m eans th at the genotype does not predict
the pattern or severity of disease.
deltaF508 mut ation is c ommon in n orthern Europeans and W1282X is common in
Ashkenazi Jews.
As a result of the mutations m entioned above, decreased quantity or functi on (and
sometimes both) of CFTR pr oteins at the epithelial cell surf ace – These defects in
CFTR pr oteins limit i on transport thr ough th e apical cell membrane, Defective ion
transport in th e lungs, p ancreas, gastrointestinal (GI) syst em, sinus es, sw eat glands,
and reproductiv e system leads to the sympt oms of Cystic Fibr osis.Th e resulting
imbalance of fluid and electrolytes causes thick, sticky mucus (in lungs, sinus es) or
viscous secretions (in p ancreas, GI tr act, reproductiv e tract) to accumul ate, which
interferes with th e proper functi on of these organs

Defective chloride ion transport in th e sweat gland leads to high s alt concentration in
sweat, but d oes not imp act the morphology of the gland.(16)

1.4. CFTR activity and ph enotype: Total CFTR activity
Total CFTR activity, which is m ainly d etermined by CFTR g enotype, is one of a
few factors that influ ence the phenotype of an individu al and determin es if h e or she
will d evelop cystic fibrosis (CF) dis ease and to what degree. The mutations present on
both CFTR alleles determin e CFTR protein producti on and activity.
Individu als with 2 n ormal (wild -type) CFTR alleles produce CFTR pr oteins of
normal quantity and functi on, and therefore, suffici ent activity. Th ese individu als
neither have nor are carriers of CF.
Carriers of CF h ave 1 normal CFTR allele, which produces normal CFTR
protein, and 1 mut ated CFTR allele, which produces defective CFTR pr otein with
reduced quantity or functi on. In this c ase, there is suffici ent functi onal CFTR protein,
and hence CFTR activity, t o result in a non-CF ph enotype. Nonetheless, some carriers
may have increased risk f or certain pulm onary conditions (e.g., asthm a).
CF-causing mut ations on both alleles

Fig 6 -CF causing mut ations on both alleles

People who have CF-causing mut ations on both alleles produce CFTR pr oteins th at
are defective in qu antity or functi on (and sometimes both), l eading t o a reducti on of
total CFTR activity and a CF ph enotype.
The degree of reducti on in total CFTR activity relates to the extent of CF
manifestations. Mut ations th at reduce but retain some residual CFTR activity c an be
associated with a variation in sympt oms and a spectrum of phenotypes.
 Individu als with 2 n ormal alleles and CF carriers have the highest levels of total
CFTR activity and no CF dis ease
 Patients with CFTR -related dis orders have intermediate levels of total CFTR activity
 Patients with CF h ave the lowest levels of total CFTR activity
Spectrum of phenotypes associated with t otal CFTR activity

Fig 7 -Spectrum of CFTR activity
CBAVD, c ongenital bilateral absence of the vas deferens.

CFTR mutations on both alleles contribut e to the quantity of functi onal CFTR pr oteins
and respective levels of total CFTR activity. CFTR mutations can result in either littl e
to no CFTR pr otein activity or in r esidual CFTR protein activity. Th e extent to which
the combin ation of CFTR alleles affects CFTR pr otein activity (i. e., normal, residual,
little to no) influ ences the phenotype of an individu al person.(17,18,19,20,21,22)
Spectrum of phenotypes associated with CFTR activity of both alleles

Fig 8 – Spectrum of CFTR activity
1.5. Clinic al presentation
Gastrointestinal tract manifestation
Meconium il eus occurs in 7 -10% of patients with cystic fibr osis. P atients with simpl e
meconium il eus usu ally pr esent with abdominal dist ension at birth, eventually
progressing t o failure to pass meconium, bili ous vomiting, and pr ogressive abdominal
distension.(23,24,25,26,27)
Patients with c omplic ated meconium il eus pr esent more dramatically at birth with
severe abdominal dist ention, sometimes accompanied by abdominal wall erythema and
edema. Abdominal dist ention may be severe enough t o cause respiratory distr ess.

Other GI m anifestations in n eonates includ e intestinal obstructi on at birth and various
surgic al findings ( eg, volvulus, int estinal atresia, perforation, meconium p eritonitis).
Less commonly, p assage of meconium m ay be delayed (>24 -48 hours after birth) or
cholestatic jaundic e may be prolonged.
Infants and childr en present with incr eased frequency of stools, which sugg ests
malabsorption (ie, fat or oil drops in st ools), failure to thriv e, intussusc eption
(ileocecal), or rectal prolapse.(28,29,30)

Fig 9 – Organs affecting in CF

Patients with p ancreatic insuffici ency h ave fat-soluble vitamin d eficiency and
malabsorption of fats, pr oteins, and carbohydrates (however, malabsorption of
carbohydrates is n ot as severe as that of fats and pr oteins). P atients pr esent with f ailure
to thriv e (despite an adequate appetite), flatulence or foul-smelling fl atus, r ecurrent
abdominal pain, and abdominal dist ention.(31,32,33,34)

Malabsorption results in st eatorrhea, characterized by fr equent, poorly formed, large,
bulky, f oul-smelling, gr easy stools that float in w ater. Cloth diapers, if us ed, are
difficult t o clean. Alternatively, some patients h ave anorexia without obvious
steatorrhea.(35,36,37)
Patients m ay present with a history of jaundic e or gastrointestinal tract bleeding as a
result of hepatobiliary inv olvement.(38,39)
Respiratory tract manifestations
Patients pr esent with a chronic or recurrent cough, which c an be dry and hacking at the
beginning and can produce mucoid (early) and purul ent (later) sputum. Prolonged
sympt oms of bronchiolitis occur in inf ants. P aroxysm al cough f ollowed by v omiting
may occur.
Recurrent wh eezing, r ecurrent pn eumonia, atypic al asthm a, pneumothorax, hemoptysis,
and digit al clubbing are all complic ations and m ay be the initial manifestation. Dyspn ea
on exertion, hist ory of chest pain, recurrent sinusitis, n asal polyps, and hemoptysis m ay
also occur.
Urogenital tract manifestations
Undescended testicles or hydr ocele may be present in b oys. M ales are frequently st erile
because of the absence of the vas deferens. Th erefore, male infertility m ay be one of
the presentations.
Fertility is m aintained, although p ossibly d ecreased, in f emales. Secondary sexual
development is often delayed. Amenorrhea may occur in f emales with s evere
nutriti onal or pulm onary inv olvement.

Other Signs and sympt oms
Other signs and sympt oms of CF are related to an ups et of the balance of min erals in
blood.
CF c auses your sw eat to become very salty. c an cause clubbing and low bone density.
NOTE-People with 2 CFTR mut ations resulting in l oss of CFTR activity generally have
a CF phenotype, which m ay include– Elevated sweat chloride (>60 mmol/L) –

Pancreatic insuffici ency – CBAVD– Lung functi on decline over time – Pseudomonas
aeruginosa colonization.
1.6. Methods of diagnosis
Sweat chloride test
The measurement of sweatरelectrolyte concentrationsरhas been the mainstay of
diagnosing CFरsince a standardized procedure, known as the Gibs on-Cooke method,
was establish ed inर1959.Subs equent analysis of isolatedरsingl e sweat ducts id entified
chloride as the principl eरelectrolyte affected in CF.Th e discovery ofरCFTR c onfirm ed
the role of electrolyte transportरin the etiology of CF and gave a molecularरrationale to
the sweat test for diagnosing CF. Although th e ability t o test for CFTR g ene mutations
gives a new dim ension toरdiagnosing CF,th e sweat chloride test remains th e
standardरprocedure to confirm a CF di agnosis. Th e sweatरtest inv olves
transdermalरadministr ation ofरpilocarpine by iontophoresis to stimul ateरsweat gland
secretion, followed by c ollection and quantitation of sweat onto gauze or filter paperरor
into a Macroduct c oilरand analysis of chloride concentration.(40,41,42,43,44)
Test Int erpretation of sweat chloride test.
Since the introducti on of theरorigin al standardized sweat test methodology,
universalरdefiniti ons of normal (≤39 mmol/L), int ermediate (40 to 59 mmol/L), and
abnormal (≥ 60रmmol/L) sw eat chloride values haveरbeen applied to all patients
regardless of age.
Most inf ants identified by N ew Born scr eening will und ergo sweat testing after 2 w eeks
of age. Earlier testing c ould lead to misleading r esults, b ecause sweat chloride

concentrations in h ealthy n ewborns gr adually decrease over the first w eeks of
life.(45,46,47).
Genotyping
Genotype testing is r ecommended for individu als with a positive family hist ory and for
couples planning a pregnancy. For those individu als with sw eat chloride values in th e
intermediate range, DN A analysis c an help establish th e diagnosis.
To be considered a cause of CF, th e mutation must:
1. Cause a change in amino acid sequence that severely affects CFTR synth esis or
functi on; or
2. Introduce a premature termin ation sign al; or
3. Alter invariant nucl eotides of intr on splic e sites; or
4. Cause a novel amino acid sequence that does not occur in th e normal CFTR g enes
from at least 100 c arriers of CF mut ations from the patient’s ethnic gr oup.(48,49,50)
Other ancilli ary tests
Ancillary tests m ay help establish a diagnosis of CF either by r evealing a phenotype,
such as Pancreatic insuffici ency, or by id entifying an ion channel abnormality.
Information regarding p ancreatic exocrine functi on is v aluable for both diagnostic and
treatment purp oses. Alternative screening t ests m easure the fecal concentration of
endogenous pancreatic enzym es. Because fecal trypsin and chym otrypsin t ests m ay be
inaccurate due to intralumin al degradation and cr oss-reactivity with ing ested enzym es,
the highly specific monoclonal test for fecal elastase, which is r esistant to degradation,
is preferred. Because of its ease of use, this t est is r ecommended for evaluating
pancreatic functi on at diagnosis and for monitoring individu als with P ancreatic
suffici ency

The Nasal Potential Difference (NPD) t est, which h as been used in CF r esearch for
decades, has recently been intr oduced to clinic al practice to aid diagnosis;74 it m ay be
particul arly helpful in individu als with inc onclusiv e sweat chloride values.75 CF is
indic ated by th e presence of a high p otential diff erence during b aseline measurements

plus a very low voltage response to zero-chloride perfusate and is oproterenol. An NPD
test showing a signific ant response to zerochloride perfusate containing is oproterenol
may be useful in ruling out a diagnosis of CF. But the quantitative aspects of NPD
results th at are clearly indic ative of CF are not defined consistently across all testing
centers.
Immun oreactive trypsin ogen (IRT) is a pancreatic enzym e that can help with
diagnosing CF in n eonates with m econium il eus wh en IRT r elative ratios are elevated
greater than the 99th p ercentile. IRT plus sw eat test was shown to increase sensitivity
and sp ecificity in scr eening.
Broncho alveolar lavage and sputum micr obiology
Airway infl ammation is th e hallmark of lung dis ease in patients with CF.
Bronchoalveolar lavage fluid usu ally sh ows a high p ercentage of neutrophils, and
recovery of Pseudomonas aeruginosa from bronchoalveolar lavage fluid supp orts th e
diagnosis of CF in a clinic ally atypic al case.(51,52,53,54,55,56,57,58)
Conclusion of chapter I
CF affects m ostly white population and its an autosomal recessive disorder.It affects
majority of the organs in th e body including lungs,p ancreas,liver and gastrointestinal
tract.More than 2000 mut ations h ave been identified and th e most common is
deletion of phenylalanine at the position of 508.Sw eat test is th e gold st andard
diagnostic m ethod and karytyping is th e best method in scr eening.

CHAPTER II. Results and Discussi ons
3.1. C omparative study b etween population of Moldova and Indi a
1 in 2000 is th e prevalence of Cystic fibr osis patient in M oldova whereas 1 in 40000
to 100000 is th e prevalence in India. More than 2000 mut ations have been identified
in CFTR g ene in diff erent ways.ΔF508, which m eans deletion of phenylalanine at the
508 p ositon, is th e most common mut ation found. Th e most frequent mut ations of the
CFTR g ene in M oldavian populations are ΔF508,G542X & W1282X, and in Indi a
ΔF508, -219insG & S169G.
3.2. Recommendation for diagnosing and scr eening
In individu als presenting with sympt oms of CF ( Fig 7 ) or a positive family hist ory,
the following di agnostic pr ocess is r ecommended:
1. A diagnosis of CF c an be made if the sweat chloride value is ≥60 mm ol/L. A
second, confirm atory sw eat chloride test is r ecommended unl ess mut ation analysis
identifies
the presence of 2 CF -causing mut ations .These patients, wh o may present
at any age, are likely to develop CF lung dis ease.
2. A sweat chloride value ≤39 mm ol/L in individu als over age 6 months is n ot
consistent with a diagnosis of CF. CF is unlik ely in this group. H owever, 2 id entified
CF-causing mutations can occur in this gr oup; th ese individu als have CF and sh ould
be followed in a CF c are center.
3. Individu als with sw eat chloride values in th e intermediate range (30 to 59 mm ol/L
for infants und er age 6 months; 40 t o 59 mm ol/L for older individu als) sh ould
undergo extensive CFTR mut ation analysis (i e, expanded panel of CFTR mut ations,
evaluation for deletions, or gene sequencing):
a. In the presence of 2 CF -causing mut ations, a diagnosis of CF c an be made.
b. Individu als with n o or 1 CF -causing mut ation and clinic al findings
sugg estive of CFTR dysfuncti on (ie, obstructiv e azoospermia, bronchiectasis, or
acute, recurrent, or chronic p ancreatitis) m ay be diagnosed with a CFTR -related

disorder, depending on their clinic al pictur e or family history, and are at risk f or CF.
Sweat chloride testing sh ould b e repeated in infants by age 2 to 6 months and
immediately in older individu als. If sw eat chloride values remain in th e intermediate
range on repeat testing, th en furth er assessment sh ould b e performed at a CF c are
center that can provide basic and ancillary testing t o clarify th e diagnosis, including:
• Clinic al assessment
• Expanded genetic testing
• Exocrine pancreatic functi on tests
• Respiratory tract cultur e for CF-associated pathogens, especially P aeruginosa.
Depending on clinic al presentation, assessment also may includ e ancillary
tests, such as:
• Genital evaluation in m ales (ie, genital examination, rectal
ultrasound, s emen analysis)
• Pancreatic im aging
• High -resolution chest CT
• Bronchoalveolar lavage, including micr obiology assessment
• Pulm onary functi on testing (n ot routinely recommended in infants at this tim e)
• NPD t esting
• Exclusi onary testing f or ciliary dyskin esia and immun e deficiency.
Signific ant clinic al signs or sympt oms of CF, l aboratory indic ation of PI, or a positive
cultur e for a CF-associated pathogen (especially P aeruginosa), should b e considered
strongly sugg estive of CF. Individu als wh o have sweat chloride values in th e
intermediate range and exhibit n o signific ant signs of CF sh ould b e monitored
periodically for the appearance of sympt oms until th e diagnosis can be ruled in or
out.
Screening of Cystic Fibr osis
Antenatal screening – Includ es Chr onic vill ous sampling(CVS) and amniocentesis to
identify abnormal CFTR g ene.

In amniocentesis, d octor inserts a hollow needle through abdominal wall into uterus
and removes a small amount of fluid fr om the sac around th e baby. Th e fluid is
tested to see whether both of the baby's CFTR genes are normal.
In CVS, d octor threads a thin tub e through th e vagina and cervix t o the placenta. The
doctor removes a tissue sample from the placenta using g entle sucti on and sample is
tested.
Cystic Fibr osis C arrier Testing -People who have one normal CFTR g ene and one
faulty CFTR g ene are CF c arriers. CF c arriers usu ally have no sympt oms of CF and
live normal lives. However, carriers can pass faulty CFTR g enes on to their
childr en.If p eople have a family hist ory of CF or a partner who has CF ( or a family
history of it),a genetics c ounselor can test a blood or saliva sample to find out
whether you have a faulty CF g ene.

3.3. Management of Cystic Fibr osis
ENHANCEMENT OF MUC OCILI ARY CL EARANCE
CF is ch aracterized by r etained dry thick mucus th at serves as a nidus f or chronic
infection. Airway clearance is considered an integral component of the management
of CF. R ecent CF pulm onary guid elines recommended that daily airway clearance
should b e provided to all patients with CF . Aerobic exercise is recommended as an
adjunctiv e therapy for airway clearance and for its additional benefits to overall
health .Because the primary initi ating event for airway obstructi on in CF is th e dry
and thick mucus, muc olytics are a logical firstlin e therapy. Th e viscid n ature of CF
sputum is l argely du e to DNA from the vast numb ers of degenerating n eutrophils
present in th e airways. Clinic al studi es showed that inhalation of recombin ant hum an
DNase (dornase alfa, or Pulm ozyme) was associated with impr oved lung functi on. .

Rehydrating agents, which r eestablish th e hydration state of the ASL by dr awing
water from the interstitium int o the Airway Surf ace Liquid vi a imposed
transepithelial osmotic gr adients, c an impr ove MCC r ates. Osmotic agents also may
stimul ate mucus cl earance by incr easing cili ary beat frequency and cough cl earance .
Inhaled hyp erosmolar agents such as hyp ertonic saline induc e osmotic flow of water
into the mucus l ayer, thereby rehydrating s ecretions and impr oving mucus rh eology
and transportability of sputum and incr easing hydr ation of the airway surf ace.
Inhalations of hyp ertonic saline (4 ml twic e daily after pretreatment with
bronchodilators) impr oved MCC and lung functi on and reduced exacerbation rates in
patients with CF. This impr ovement in muc ociliary functi on may reduce bacterial
load and chr onic infl ammation within th e airways with a concomitant stabilization of
lung functi on.
PREVENTION OF INF ECTION WITH P. AERUGIN OSA
Respiratory inf ection with P. aerugin osa is well recognized as a leading c ause of
morbidity and m ortality in p atients with CF. Th e presence of P. aerugin osa in the
lower airways is associated with a more rapid d ecline in pulm onary functi on,
worsening nutriti onal status, m ore hospital admissi ons, and a shorter life
expectancy.One of the keys to successful m anagement of CF is t o prevent inf ection
with P. aerugin osa. t a 28-day regimen of inhaled tobramycin (300 mg b.i.d.) is
effective in treating early P. aerugin osa infection .No therapy w as shown to be
effective in preventing P. aerugin osa colonization. M ethicillin -resistant S. aureus
(MRS A) is an imp ortant emerging p athogen in CF. Th e prevalence of MRS A in the
respiratory tract of individu als with CF h as incr eased subst antially, and according t o
one study it is n ow more than 20%. MRS A is associated with w orse surviv al, and
aggressive treatment aimed to eradicate MRS A should b e considered.
TREATMENT OF AIRW AY INFL AMMATION
Airway infl ammation in p atients with CF b egins early in lif e and results in incr eased
airway obstructi on and pr ogressive damage. An exaggerated infl ammatory response,

relative to the burden of infection, is r esponsibl e for much of the pathology found in
the CF lungs .The respiratory syst em in CF is ch aracterized by high c oncentrations of
neutrophils and pr oinflammatory cytokines with r educed concentrations of
antiinfl ammatory factors .
Macrolides, mainly azithromycin, are being us ed to reduce inflammation in p atients
with CF c olonized with P. aerugin osa. Azithromycin giv en orally 3 tim es a week, 250
mg (b elow 36 kg of weight) or 500 mg (m ore than 36 kg of weight) w as shown to
signific antly r educe the numb er of respiratory exacerbations and th e rate in decline of
lung functi on as well as impr oving qu ality of life .It can also reduce sputum visc osity
and airway adhesion of P. aerugin osa and disrupt th e ability of the bacteria to produce
alginate . Recently, azithromycin w as also associated with a signific ant reducti on in
pulm onary exacerbations and a signific ant incr ease in weight g ain in p atients n ot
infected with P. aerugin osa .
MAINTENANCE OF GOOD NUTRITI ON AND ENERGY B ALANCE
In most patients with CF, th e earliest manifestations of the disease are related to its
associated gastrointestinal and nutriti onal derangements. D estructi on of acinar
pancreatic tissu e, pancreatic ductul ar obstructi on, and lack of enzym atic activity l ead
to malabsorption (particul arly of fats), di arrhea, and failure to thriv e. Progressive
lung dis ease furth er incr eases calorie requirements by incr easing th e work of
breathing. th e use of pancreatic enzym e replacement therapy with emphasis on high –
calorie, high -protein, unr estrict ed diet and pr evention of fat-soluble vitamin
deficiency. Nutriti onal status sh ould b e monitored closely during r outine visits t o
allow for early int ervention once derangements are noted. In addition, the annual
nutriti onal assessment sh ould optimally includ e body composition, bone density,
glucose tolerance, and various bi ochemical and micr onutri ent levels .Early and
aggressive nutriti onal supp ort with adequate pancreatic replacement management
should lead to both normal growth and better lung functi on
REGUL AR EVALUATION OF TH E HEALTH ST ATUS

Centered care by a dedicated team of trained and experienced health pr ofessionals is
essential for optimal patient management and outcome. Care involves frequent
clinic al evaluations and m onitoring f or complic ations by physici ans and other
healthcare workers specifically tr ained in th e management of CF .
Conclusion of chapter II
Signific ant impr ovement in th e surviv al of patients with CF h as been achieved in th e
last decades. Th e impr oved clinic al status of the patients is m ainly th e result of a
better und erstanding of the natural course of infection and infl ammation in CF th at
has led to the implementation of strategies that incr ease the life expectancy and
quality of life of the patients. Th ese strategies includ e prompt di agnosis, tim ely and
aggressive nutriti onal supp ort, augmentation of MCC and impr oved muc ous
drainage, initi ation of antimicr obial and antiinfl ammatory therapy as soon as
possible, early tr eatment of acute exacerbations, impl ementation of effective hygienic
measures in and outsid e CF c enters, and pr ompt id entific ation and treatment of CF-
related complic ations. Tr eatment at a specialized CF c enter by a multidisciplin ary
dedicated team, including fr equent visits, and periodic routine tests are essential to
detect and treat early ch anges. Adherence to these therapies is ch allenging and it
should b e discuss ed with th e patients at every clinic visit. Cr eating th erapeutic
alliance with th e patients and th eir families is essential to promoting p atients’
empowerment and supp orting b etter adherence to therapies. Maintaining p atients in
optimal status will allow them to benefit from futur e treatments d esigned to correct or
modify th e basic genetic defect associated with CFTR by g ene replacement therapy
or pharmacological interventions curr ently und er development. Th ese new therapies
are expected to furth er incr ease life expectancy of the patients.

GENERAL CONCLUSI ON
1 in 2000 is th e prevalence of Cystic fibr osis patient in M oldova whereas 1 in 40000
to 100000 is th e prevalence in Indi a.Recent statistics sugg est that 1 in 25000
expatriates of Indi a in Unit ed Kingd om and Unit ed States of America have Cystic
fibrosis. H owever, the exact numb er of Cystic Fibr osis patients in Indi a are unkn own
compared to Moldova due to the lack of studi es conduct ed in th e Indian population
and also non availability of screening or investigation methods.
More than 2000 mut ations have been identified in CFTR g ene in diff erent
ways.ΔF508, which m eans deletion of phenylalanine at the 508 p ositon, is th e most
common mut ation found. Th e most frequent mut ations of the CFTR g ene in
Moldavian populations are ΔF508,G542X & W1282X, and in Indi a ΔF508, –
219insG & S169G.
Better und erstanding and scr eening of the population have increased the life
expectancy of the cystic fibr osis patients. N ew scr eening m ethods need to be
implemented into the health care systems as well as holding s eminars for the health
care professionals to improve the diagnosis and patient supp ort. Early di agnosis will
impr ove the life of patient and reduce mortality.

BIBLI OGRAFIE

DECLARAȚIE
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prezentată nici odată la o altă facultate sau instituți e de învățământ sup erior din ț ară
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limbă, sunt scris e între ghilim ele și dețin referința precisă a sursei;
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precisă;
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08.04 .2016
Absolvent: Pascal Maria

________________________
(semnătur a în origin al)