slidesA slidesB

Web sites too many to list, but a good start is

OMIM ENTRY for CF    official, up to date, complete
CF-I       the Cystic Fibrosis Web Ring
CF-II    another individual page with many links
CF-III
Cystic Fibrosis 101

I. INTRODUCTION

Cystic Fibrosis (CF) is the most common, severe autosomal recessive disorder in the Caucasian population
~5% of North Americans are carriers; they are completely asymptomatic
1/2000 live births are affected by CF
                               (q = 0.022; p = 0.978; 2pq = 0.043 (~5%))
there are an estimated 30,000 CF patients in the US
carrier identification and counseling could make a major impact on the incidence of the disease
significant numbers of affected individuals among African Americans (1/17000); only 1/90000 in Asians
- the carrier frequency varies among ethnic groups

physiology: first clinical description in 1938: defect affects certain cells of the body:
                   secretory cells of the pancreas (fibrotic lesions detected)
                   sweat glands
                   epithelial cells lining the respiratory tract
                   functions of the male reproductive system

i.e. defective/abnormal exocrine secretions

symptoms:
digestive problems - pancreatic insufficiency corrected by enzyme supplements in the diet
high concentration of electrolytes in perspiration - the saltiness is a minor problem, but used conveniently in diagnosis
dehydrated mucus in respiratory tract - thicker, stickier, difficult to clear, hence susceptible to infections (Pseudomonas species) leading to lung damage, and death on average at age 27.
5 - 10% of CF newborns have intestinal obstructions called meconium ileus
males are frequently sterile (absence or obstruction of vas deferens)

not much of a clue as to the biochemical defect, and much time was spent on efforts to define the lesion biochemically (until ~ 1990);      at one point there was a guess that the problem was with the cilia of the epithelium lining the respiratory tract: bodily fluids of CF patients were tested with cilia from the gills of mussels to observe inhibition of ciliary movement

the hope/goal was to define an enzyme (or inhibitor) which can be quantitated in normal and heterozygous individuals to identify the carriers

modern treatment:
i) physical therapy to loosen and release mucus; chest percussion
ii) antibiotics to treat infections

II. GENETICS

autosomal, recessive;
carriers completely asymptomatic;
no large deletions or chromosome rearrangements known to be associated with CF, as in the case of DMD
why is it so frequent? why is its frequency so dependent on the subpopulation, i.e. much lower incidence in Hawaiian population?
in the case of DMD the explanation is in part that the gene and hence the target is so large
more importantly, there are tandem repeats which can lead to unequal crossing over during meiosis, leading to duplications and deletions (similar to what is observed with the opsin genes on the X chromosome)
- 1/3 of the DMD mutations are spontaneous

most recent hypothesis: CF heterozygotes may be more resistant to cholera toxin (rationale to become apparent)

after a lot of work involving CF pedigrees: linkage analysis based on large number of RFLPs assigned the CF locus to the long arm of chromosome 7, band q31
- two closely linked flanking markers were found: the oncogene MET, and the anonymous sequence D7S8 (within 1 centimorgan or 1000-1500 kb)

....MET........CF.........D7S8...

III. GENE CLONING

positional cloning - dependent on accurate mapping and reasonable estimate of physical distance from recombination frequencies
- two or three labs in Toronto and Ann Arbor: Lap-Chee Tsui, F.S. Collins et al (13 or more) - success in 1989 cost: estimated $15 Mill.

1. saturation mapping approach; brute force approach; very expensive, but justified by the high incidence of the disease and the prospects of carrier identification
systematic examination of cloned markers from somatic cell hybrids
ten genomic librairies were constructed and screened
flow sorting of chromosome 7 to make libraries
cDNA libraries from cultured sweat gland cells of a non CF individual
chromosome walking and chromosome jumping
a continuous region of 280 kb was mapped and covered by overlapping clones
pulsed field gel electrophoresis to establish physical linkage
over short intervals, a special technique used most successfully here: "jumping clones"
- another promising technique for cloning from a cytologically defined locus: microdisection of chromosomes [see Nature 338:348- (1989) ]

2. a major problem: given a segment of cloned DNA (from a total segment of 1000-2000kb), how does one know whether it is a gene, and whether it is the desired gene?
criteria:
i) cross-hybridizing sequences (on Zoo blots-similar to ZFY gene identification on Y chromosome)
ii) identification of CpG islands, common at 5' ends of vertebrate genes
iii) examination of possible mRNAs from tissues affected in CF patients
iv) examination of cDNA sequences from selected tissues
v) sequencing and identification of ORFs: four regions were identified, and three were eliminated as CF candidates by genetic and DNA sequence analysis

3. cloning of complete cDNA with the help of one probe believed to be in the CF locus
- several cDNA libraries from sweat gland cells, lung, pancreas, lung were screened and 18 clones were isolated
- clones were overlapping
- transcript (mRNA) detected was ~6.5 kb; transcripts found in pancreas, sweat glands, nasal polyps, lung, liver, but not in skin fibroblasts, lymphocytes, brain
- defined genomic region of ~250 kb; there are 27 exons
- sequence gave ORF with 1480 aa

IV. THE PROTEIN ENCODED BY THE CF LOCUS

nucleotide sequence of cDNA: 1480 aa --> 168,138 kD protein

12 membrane-spanning domains;
N-terminal and C-terminal on cytoplasmic side of plasma membrane;
N-linked carbohydrate on external domain;
two ATP binding domains;
highly charged cytoplasmic domain - R-domain - defined by single large exon: has 9 sites (consensus) for phosphorylation by protein kinase A, and seven for phosphorylation by protein kinase C.

relationship to other cation channel proteins
relationship to mdr (multidrug resistance P-glycoprotein) (see Juranka, Zastawny, and Ling FASEB J. 3:2583-2592 for a review)
- relationship to transport systems of bacteria and other organisms
- belongs to a superfamily of ABC transport proteins (ATP binding casette)

CF is a transmembrane conductance regulator (CFTR)

this agrees with electrophysiological measurements and comparisons of normal and CF preparations

Questions:
i) is it the ion channel itself, or does it regulate an ion channel?
ii) is ATP hydrolysis involved in a transport function (i.e. is it an active transporter)? expression of the gene in other cells confirmed the function of the protein

More recent studies on the function of the CFTR: correction of chloride channel defect in transfected cells: mouse fibroblasts, CHO cells, HeLa cells, Xenopus oocytes

chloride conductance activated by cAMP;
cAMP stimulates PKA which phosphorylates R domain

now established: CFTR can function as an ion channel, activatable by PKA, but may have other functions as well - defective acidification of intracellular organelles of CF patients - could account for abnormal sialation and sulfation of glycoproteins - could account for defective endo- and exocytosis

CFTR in cell-free membrane patches: phosphorylation of the R domain and ATP binding are required to open the chloride channel: there is no chloride pumping against a gradient, driven by ATP hydrolysis

ATP hydrolysis may be needed once to open the channel OR: ATP when bound, keeps the channel open, but is hydrolysed to close the channel again (similar to G-protein function)

latest insights: ATP hydrolysis at NBF1 converts the channel from inactive to active closed; binding of ATP at NBF2 opens the channel
ATP hydrolysis at NBF2 closes the channel - when a nonhydrolyzable ATP analog is bound to NBF2 the channel stays open (TIBS 19:513-518, 1994; Cell 82: 231-239, 1995)

lots of studies on role of mutations on activity of CFTR: some mutations alter protein processing, i.e. glycosylation in ER/Golgi, which could be the primary defect in many CF mutations

V. ANALYSIS OF THE CF GENE IN CF PATIENTS

(i.e. we have identified a gene encoding a gene product which makes some sense, but it still remains to be proven that this is the CF locus)

- CFTR mRNA was found in sweat glands of CF patients, i.e in contrast to DMD there are no mutations changing the size of the transcript
- genetic evidence favors one locus
- detection of allelic and haplotype association between CF and closely linked RFLPs suggest that a single mutational event may account for most of the CF mutations in the Northern European population
- a second mutation may be prevalent in some Southern European populations

- the lack of a well-developed functional assay for the CF gene product required a detailed examination of the CF locus and comparison between CF and normal alleles
- an antibody does not help, if the mutant protein is present at normal levels in the affected individuals
- only parents of CF patients, each of whom by definition carries an N and a CF chromosome, were suitable for the analysis
- one has to distinguish between polymorphisms in the gene which have no consequence and the true mutations

2. RFLPs and family studies: localize recombination breakpoints
CEN7.....MET... JG2E1. CF..D7S424..............TEL

3. Allelic association (or linkage disequilibrium): the more closely linked the markers, the greater the likelyhood that linkage equilibrium has not been established
additional confirmation of identification of CF locus: strong association with CF for the closer markers, little or no association detected for the more distant markers on either side

4. Nucleotide sequence comparisons in CF gene of normal and CF patients
general survey of CF patients and normal individuals: amplify the region in question by PCR probe with sequence-specific oligonucleotide probes
68% of the CF patient population had the mutation DeltaPhe508, a 3 bp deletion which leads to the deletion of phenylalanine 508
this mutation is located in one of the potential ATP binding domains (exon 10)
none of the normal chromosomes had this deletion
guess: strong founder effect for this deletion or a significant heterozygote selection in Northern European population (cholera??)

5. Implications for genetic diagnosis
1. why the high frequency?
a. high mutation rate ?
b. heterozygote advantage ? Science 266:107 (1994): CF heterozygote resistance to cholera toxin in the CF mouse model
c. multiple loci ?
d. reproductive compensation ?
by now a total of >170 mutations have been described in the CFTR gene; 20 are common, the rest are rare - the frequency of common mutations varies with geography and ethnic groups
data accumulated by the Cystic Fibrosis Genetic Analysis Consortium, a network of >88 labs around the world several types of CF patients exist, with different severity of the disease and hence clinical courses
the mildest form: infertile males;
about 85 to 90% of CF carriers can in principle now be detected

QUESTIONS:

i) should one go ahead on such a large scale screening process?
ii) can one counsel such a large number of potential carriers?
iii) how will people react to statistics and the prediction of a variable course of the disease?
iv) allele frequencies

Example: CF-PI: pancreatic-insufficient CF-PS: pancreatic-sufficient

hypothesis: there are various mutant alleles of the CF locus, which can be subdivided into severe (S) and mild (M) alleles
if S is recessive to M, then one can estimate that of all mutated CF alleles, the mild mutations represent 8% (0.08), and the severe mutations represent 92% (0.92)
the DeltaPhe508 mutation belongs to the S alleles and represents 68% of all CF mutations, hence the remainder of the severe mutations make up 24% (92-68)

CF-PS patients can be compound heterozygotes, i.e. carry two different mutant alleles
the predicted and observed frequencies are in general agreement with each other (CF alleles among the CF population are distributed according to the Hardy-Weinberg law)

with such a large locus, why are there no deletions or translocation-breakpoints observed among the CF patients?
- it may be that only relatively few specific alterations in the CF locus can be tolerated to give a live birth
- a more serious disruption or deletion of the gene may be embryonic lethal

7. Prospects for Gene Therapy
i) relevant cells to treat? airway lining cells; submucosal glands
ii) what fraction must be corrected? as few as 6% (if gap junctions exist between cells in an epithelium)
iii) is overexpression toxic? not in transgenic mice
iv) how long will expression persist? integrating vectors may be mutagenic;
v) will there be adverse immune responses?
vi) other safety considerations???

see F. Collins (1992) Science 256:774-779

Selected References

A. Historical

- Anderson, M.P., Gregory, R.J., Thompson, S., Souza, D.W., Paul, S., Mulligan, R.C., Smith, A.E., and Welsh, M.J. (1991) Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253, 202-205.
- Caskey, C.T., Kaback, M.M., and Beaudet, A.L. (1990). The American Society of Human Genetics statement on cystic fibrosis screening. Am. J. Hum. Genet. 46, 393-393.
- Goodfellow, P.N. (1989). Cystic fibrosis: Steady steps lead to the gene. Nature 341, 102-103.
- Gorden, P. (1991). Cystic fibrosis research. Science 251, 500.
- Higgins, C.F. and Hyde, S.C. (1991) Cystic fibrosis: Channelling our thoughts. Nature 352, 194-195
- Kerem, B., Rommens, J.M., Buchanan, J.A., Markiewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., and Tsui, L.-C. (1989). Identification of the cystic fibrosis gene: Genetic analysis. Science 245, 1073-1080.
- Levitan, I.B. (1989). The basic defect in cystic fibrosis. Science 244, 1423-1423.
- Marx, J.L. (1989). The CF gene hits the news. Science 245, 924-924.
- Ringe, D. and Petsko, G.A. (1990). Cystic fibrosis: A transport problem. Nature 346, 312-313.
- Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M.L., Iannuzzi, M.C., Collins, F.S., and Tsui, L.-C. (1989). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066-1073.
- Rommens, J.M., Iannuzzi, M.C., Kerem, B., Drumm, M.L., Melmer, G., Dean, M., Rozmahel, R., Cole, J.L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J.R., Tsui, L.-C., and Collins, F.S. (1989). Identification of the cystic fibrosis gene: Chromosome walking and jumping. Science 245, 1059-1065.
- Stewart, A.D. (1989). Screening for cystic fibrosis. Nature 341, 696-696.
- Trapnell, B.C., Chu, C.-S., Paakko, P.K., Banks, T.C., Yoshimura, K., Ferrans, V.J., Chernick, M.S., Crystal, R.G. (1991). Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis. PNAS 88, 6565-6569.
- Tsui, L.-C. and Buchwald, M. (1991). Biochemical and molecular genetics of cystic fibrosis. Adv. Hum. Genet. 20, 153-266.
-Tsui, L.-C. (1992). The spectrum of cystic fibrosis mutations. Trends Genet. 8, 392-398.
-Tsui, L.-C. and Buchwald, M. (1991). Biochemical and molecular genetics of cystic fibrosis. Adv. Hum. Genet. 20, 153-266.

B. Recent
- Alton, E. and Geddes, D. (1994). A mixed message for cystic fibrosis gene therapy. Nature Genet. 8, 8-9.
- Gadsby, D.C. and Nairn, A.C. (1994). Regulation of CFTR channel gating. Trends Biochem. Sci. 19, 513-518.
- Grubb, B.R., Pickles, R.J., Ye, H., Yankaskas, J.R., Vick, R.N., Engelhardt, J.F., Wilson, J.M., Johnson, L.G., and Boucher, R.C. (1994). Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans. Nature 371, 802-806.
- Morral, N., Bertranpetit, J., Estivill, X., Nunes, V., Casals, T., Giménez, J., Reis, A., Varon-Mateeva, R., Macek, M., Jr., Kalaydjieva, L., Angelicheva, D., Dancheva, R., Romeo, G., Russo, M.P., Garnerone, S., Restagno, G., Ferrari, M., Magnani, C., Claustres, M., Desgeorges, M., Schwartz, M., Schwarz, M., Dallapiccola, B., and Novelli, G. (1994). The origin of the major cystic fibrosis mutation (DeltaF508) in European populations. Nature Genet. 7, 169-175.
- Reisin, I.L., Prat, A.G., Abraham, E.H., Amara, J.F., Gregory, R.J., Ausiello, D.A., and Cantiello, H.F. (1994). The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J. Biol. Chem. 269, 20584-20591.