CA2073441C - Introns and exons of the cystic fibrosis gene and mutations at various positions of the gene - Google Patents

Abstract

The cystic fibrosis gene and its gene product are described for mutant forms.
The genetic and protein information is used in developing DNA diagnosis, protein diagnosis, carrier and patient screening, cloning of the gene and manufacture of the pro-tein, and development of cystic fibrosis affected animals.

Description

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The present invention relates generally to the cystic fibrosis (CF) gene, and, more particularly to the identification, isolation and cloning of the DNA sequence corresponding to mutants of the CF gene, as well as their transcripts, gene products and genetic information at exon/intron boundaries. The, present invention also relates to methods of screening for and detection of eF
carriers, CF diagnosis, prenatal CF screening and diagnosis, and gene therapy utilizing recombinant technologies and drug therapy using the information derived from the DNA, protein, and the metabolic function of the protein.
83~rCRGRO1~1D of 'fF~IE Tl~EId'1°TON
Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the Caucasian population. It affects approximately 1 in 2000 live births in Narth America (Boat et al, The Metabniir. ua~is of Tnhgrited DiRp~~r~
6th ed, pp 2649-2680, McGraw Hill, NY (1989)). Approximately 1 in 20 persons are carriers of the disease.
Although the disease was first described in the late 193o~s, the basic defect remains unknown. The major symptoms of cystic fibrosis include chronic pulmanary disease, pancreatic exocrine insufficiency, and elevated sweat electrolyte levels. The symptoms are consistent with cystic fibrosis being an exocrine disorder.
3o Although recent advances have been made in the analysis of ion transport across the apical membrane of the epithelium of CF patient cells, it is not clear that the abnormal regulation of chloride channels represents the primary defect in the disease. Given the lack of understanding of the molecular mechanism of the disease, an alternative approach has therefore been taken in an attempt to understand the nature of the molecular defect 'WO 91/1a734 PC.T/~CA911OQ009 through direct cloning of the responsible gene on the basis of its chromosomal location.
However, there is no cleax phenotype that directs an approach to the exact nature of the genetic basis of the disease, or that allows for an identification of the cystic fibrosis gene. The nature of the CF defect in relation to the population genetics data has not been readily apparent. Both the prevalence of the disease and the clinical heterogeneity have been explained by several different mechanisms: high mutation rate, heterozygote advantage, genetic drift, multiple loci; and reproductive compensation.
Many of the hypotheses can not be tested due to the lack of knowledge of the'basic defect. Therefore, alternative approaches to the determination and characterization of the CF gene have focused on an attempt to identify the location of the gene by genetic analysis.
Linkage analysis of the CF gene to antigsnic and protein markers was attempted in the 1950'x, but no posit'ave results were obtained [Steinberg et al ~T.
FIum<, Genet. 8: 162-176, (I956); St~inberg and Morton Am.
J. Hum. Genet 8: 177-189, (1956); Goodchild et al J. Med.
G a 7: 417-419, 1976.
More recently, it has become possible to use RFLP's to facilitate linkage analysis. The first linkage of an RFLP marker to the CF gene was disclosed in 1985 [Tsui et al. ciencg 230: 1054-1057, 1985) in which linkage was found between °the CF gene and an uncharacterized marker DOCRI-917. The association was found in an analysis of 39 families with affected CF children. This showed that although the chromosomal location had not been established; the location of the disease gene had been narrowed to about 1% of the human genome, or about 30 million nucleotide base pairs.
The chromosomal location caf the DOCRI-917 probe: was established using rodent-human hybrid cell lines containing different human chromosome complements. It was shown that DOCR1-917 (and therefore the CF gene) maps to human chromosome 7.
Further physical and genetic linkage studies were pursued in an attempt to pinpoint the location of the CF gene. Zengerling et al [Am. J. Hum. Genet. 40:

236 (1987)] describe the use of human-mouse somatic cell hybrids to obtain a more detailed physical relationship between the CF gene and the markers known to be linked with it. This publication shows that the CF gene can be assigned to either the distal region of band q22 or the proximal region of band q31 on chromosome 7.
Rommens et al [Am. J. Hum. Genet. 43: 645-663, (1988)] give a detailed discussion of the isolation of many new 7q31 probes. The approach outlined led to the isolation of two new probes, D7S122 and D7S340, which are close to each other.
Pulsed field gel electrophoresis mapping indicates that these two RFLP markers are between two markers known to flank the CF gene, MET [White, R., Woodward S., Leppert M., et al. Nature 318: 382-384, (1985)] and D7S8 [Wainwright, B. J., Scambler, P. J., and J. Schmidtke, Nature 318: 384-385 (1985)], therefore in the CF
gene region. The discovery of these markers provides a starting point for chromosome walking and jumping.
Estivill et al, [Nature 326: 840-845(1987)] disclose that a candidate cDNA
gene was located and partially characterized. This however, does not teach the correct location of the CF gene. The reference discloses a candidate cDNA gene downstream of a CpG island, which are undermethylated GC nucleotide-rich regions upstream of many vertebrate genes. The chromosomal localization of the candidate locus is identified as the XV2C region. However, that actual region does not include !V0 91/10731 PC1'/CA91l00009 A major difficulty in identifying the CF gene has been the lack of cytologically detectable chromosome rearrangements or deletions, which greatly facilitated all previous successes in the cloning of human disease genes by knowledge of map position.
Such rearrangements and deletions could be observed cytologically and as a result, a physical location on a particular chromosome could be correlated with the particular disease. Further, this cytological location could be correlated with a molecular ,location based on known relationship between publicly available DNA probes and cytologically visible alterations in the chromosomes.
Knowledge of the molecular location of the gene for a particular disease would allow cloning and sequencing of that gene by routine procedures, particularly when the gene product is known and Toning success can be confirmed by immunoassay of e}:pression products of the cloned genes, Tn contrast, neither the cytological location nor the gene product of the gene for cystic fibrosis was known in the prior art. With the recent identification of MET and D7S8, markers which flanked the CF gene but did nit pinpoint its molecular location, the present inventars devised various novel gene cloning strategies to approach the CF gene in accordance with the present invention. The methods employed in these strategies include chromosome jumping from the flanking markers, cloning of DrIA fragments from a defined physical region with the use of pulsed field gel electrophoresis, a combination of somatic cell hybrid and molecular Cloning techniques designed to isolate 1~NA fragments from undermethylated CpG islands near CF, chromosome microclissection and cloning, and saturation cloning of a large number of DNA markers from the 7q31 region. By means of these novel strategies, the present inventors were able to identify the gene responsible for cystic fibrosis where the prior art was uncertaW or, even m one case, wrong.
The application of these genetic and molecular cloning strategies has allowed 5 the isolation and cDNA cloning of the cystic fibrosis gene on the basis of its chromosomal location, without the benefit of genomic rearrangements to point the way. The identification of the normal and mutant forms of the CF gene and gene products has allowed for the development of screening and diagnostic tests for CF
utilizing nucleic acid probes and antibodies to the gene product. Through interaction with the defective gene product and the pathway in which this gene product is involved, therapy through normal gene product supplementation and gene manipulation and delivery are now made possible.
The gene involved in the cystic fibrosis disease process, hereinafter the "CF
gene" and its functional equivalents, has been identified, isolated and cDNA
cloned, and its transcripts and gene products identified and sequenced. A three base pair deletion leading to the omission of a phenylalanine residue in the gene product has been determined to correspond to the mutations of the CF gene in approximately 70%
of the patients affected with CF, with different mutations involved in most if not all the remaining cases. This subject matter is disclosed in co-pending United States patent 5,776,677.
Specific aspects of the invention defined in these U.S. patent applications are discussed in journal articles by the inventors, namely, Science (1989) 245 No.

pp 1066-1073 and Science (198~~) 245 No. 4922 pp 1073-1080. These journal articles discuss the CFTR gene sequence and the mutation claimed in the U.S. patent W~ 91/10734 ~.~ ~ ~ (1 ~~ PC.°f/CA91/00009 With the identification and sequencing of the mutant gene and its gene product, nucleic acid probes and antibodies raised to the mutant gene product can be used in a variety of hybridization and immunological assays to screen for and detect the presence of either the defective CF gene or gene product. Assay kits for such screening and diagnosis can also be provided. The genetic information derived from the intron/exon boundaries is also very useful in various screening and to diagnosis procedures.
Patient therapy through supplementation with the normal gene product, whose production can be amplified using genetic and recombinant techniques, or its functional equivalent, is now also possible. Correction or modification of the defective gene product through drug treatment moans is now possible. In addition, cystic fibrosis can be cured or controlled through gene therapy by correcting the gene defect ,~ situ or using recombinant or other vehiclet~ to deliver a DNA sequence capable of expression of the normal gene product to the cells of the patient.
According to another aspect of the invention, a purified mutant CF gene comprises a DNA sequence encoding an amino acid sequence for a protein where the pxotein, when expressed in cells of the human body, is associated with altered cell function which correlates with the genetic disease cystic fibrosis.
According to another aspect of the invention, a purified RNA molecule comprises an RNA sequence corresponding to the above DNA sequence.
According to another aspect of the invention, a DNA
molecule comprises a cDNA molecule corresponding to the above DNA sequence.
According to another aspect of the invention, a.DNA
anolecule comprises a DNA sequence encoding mutant CFTR
polypeptide having the sequenc.~a according to the following Figure 1 for amino acid residue positions 1 to W~O 91/10734 PCT>CA91100009 7 '~~~.y~~'~~i w.~ L _ 1480 as further characterized by a nucleotide sequence variants resulting in deletion or alteration of amino acids or residue positions 85, 148, 378, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 arid 1092.
According to another aspect of the invention, a DNA
molecule comprises an intronless DNA sequence encoding a mutant CFTR polypeptide having the sequence according to FIgure 1 for DNA sequence positions Z to 4575 and, further characterized by nucleotide sequence variants resulting in deletion or alteration of DNA at DNA
sequence positions 129, 556, 621+1, 713.+1, 1717-1 and 3659.
According to anather aspect of the invention, a DNA
molecule comprises a cDNA molecule corresponding to the above DNA sequence.
According to another aspect of the invention, the cDNA.molecule comprises a DNA sequence selected from the group consisting of:
(a) DNA sequences which correspond to the mutant 2o DNA sequence selected from the group of mutant amino acid positions Of 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 109? and mutant pNA seqnece positions 129, 556, 621+1, 711+1, 1717-1 and 3659 and which encode, on expression, for mutant CFTR polypeptide;
(b) DNA sequences which correspond to a fragment of the selected mutant DNA sequence, including at least twenty nucleotides;
(c) DNA sequences which comprise at least twenty nucleotides and encode a fragm~:nt of the selected mutant 3o CFTR protein amino acid sequence;
(d) DNA sequences encoding an epitope encoded by at least eighteen sequential nucleotides in the selected mutant DNA sequence.
According to another aspect of the invention, a DNA
sequence selected from the group consisting of:

WO 91/10734 ~ ~ PC"~'/CA91/0~009 s (a) DNA sequences which correspond to portions of DNA sequences of boundaries of exons/introns of the genomic CF gene;
(b) DNA sequences of at least eighteen sequential nucleotides at boundaries of exons/introns of the genomic CF gene depicted in Figure 18; and (c) DNA sequences of at least eighteen sequential nucleotides of intron portions of the genomic CF gene of Figure 18.
1o According to another aspect of the invention, a purified nucleic acid probe comprises a DNA or ~tNA
nucleotide sequence corresponding to the above noted selected DNA sequences of groups (a) to (c).
According to another aspect of the invention, purified RNA molecule comprising RNA sequence corres-ponds to the mutant DNA sequence selected from the group of mutant protein positions consisting of 85, 148, 178, 455, 493, 507, 542, 549,, 551, 560, 563, 574, 1077 and 1092 and of mutant DNA sequen~a positions consisting of 129, 556, 621+1, 711+1, 1717-1 and 3659.
A purified nucleic acid probe comprising a DNA or RNA nucleotide sequence corresponding to the mutant sequences of the above recited group.
According to an~ther aspect of the invention, a recombinant cloning vector comprising the DNA sequences of the mutant DNA and fragments thereof selected from the group of mutant protein positions consisting of 85, 148, 178, 455, 493, 507, 542, 549, 551, 563, 574, 1077 arid 1092 and selected from the group of mutant DNA sequence positions consisting of 129, 556, 621+1, 711+1, 1717-1 and 3659 is provided. The vec~t:or, according to an aspect of this invention, is operatively linked to an expression control sequence in the recombinant DNA molecule so that the selected mutant DNA sequences for the mutant CFTR
polypeptide can be expressed. The expression control sequence is selected from the w~roup consisting of sequences that control the exp~:ession of genes of wo 9mo~~a Pcre~a~gnoooo9 L~~r~~~,~e~i.3'.
prokaryotic or eukaryotic cells and their viruses and combinations thereof.
According to another aspect of the inventian, a method for producing a mutant CFTR polypeptide comprises the steps of:
(a) culturing a host cell transfected with the recombinant vector for the mutant DNA sequence in a medium and under conditions favorable for expression of the mutant CFTR polypeptide selected from the group of mutant CFTR polypeptides at mutant protein positions 85, 148, 1?$, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092 and mutant DNA sequence positions 129, 556, 621+1, 711-1 1717-1 and .3659; and (b) isolating the expressed mutant CFTR
polypeptide.
According to another aspect of the invention, a purified protein of human cell membrane origin comprises an amino acid sequence ~:ncoded by the mutant DNA
sequences selected from the group of mutant protein positions of 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092 and from the group of mutant DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659 where the protein, when present in human cell membrane, is associated with cell function which causes the genetic disease cystic fibrosis.
According tc~ another aspect of the invention, a method is provided for screening a subject to determine if the subject is a CF carrier or a CF patient comprising the steps of providing a biological sample of the subject 3o to be screened and providing an assay for detecting in the biological sazaple, the presence of at least a member from the group consisting of:
(a) mutant CF gene selected from the group of mutant protein positions 85, 14$, 17$, 455, 493, 507, 542, 549, 551, 560, 563, 574, ~L077 and 1092 and from the group of mutant DNA

W~ 91 /10734 PCf/CrS91 /00009 ~~'~'~4~
sequence positions 129, 556, 621+1, 711°~1, 1717-1 and 3659;
(b) mutant CF gene products and mixtures thereof;
(c) DNA sequences which correspond to portions of 5 DNA sequences of boundaries of exons/introns of the genomic CF gene;
(d) DNA sequences of at least eighteen sequential nucleotides at boundaries of exons/introns of the genomic CF gene depicted in Figure 18; and 10 (e) DNA sequences of at least eighteen sequential nucleotides of intron portions of the genomic CF gene of Figure 18.
According to another aspect of the invention, a kit for assaying for the presence of a CF gene by immunoassay techniques comprises:
(a) an antibody which specifically binds to a gene product of the mutant DNA sequence selected from the group of mutant protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092 and from the group of mutant DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659;
(b) reagent means for detecting the binding of the antibody to the gene product; and (c) the antibody and reagent means each being present in amounts effective to perform the immunoassay.
According to another aspect of the invention, a kit fer assaying for the presence of a mutant CF gene by hybridization technique comprises:
(a) an oligonucleotide probe which specifically.
binds to the mutant CF gene having a mutz~tion at a protein position selected from the group consisting of 85, 148, 178, 455, 493, 507 , 542, 549, 551, 560, 563, 574, 1077 and 1092 or having a mutation at a DNA sequence position selected from the group consisting of 129, 556, 621+1, 711+1, 1717-1 and 3659;
(b) reagent means for detecting the hybridization of the oligonucleotide probe to the mutant CF gene; and WO 91/1073 PC'T/CA91/00009 ~, ~ '°7. c..~ i , (c) the probe and reagent means eac2i"~ing present in amounts effective to perform the hybridization assay.
According to another aspect of the invention, ari animal comprises an heterologous cell system. The cell system includes a recombinant cloning vector which includes the recombinant DNA sequence corresponding to the mutant DNA sequence which induces cystic fibrosis symptoms in the animal.
According to anpther aspect of the invention, in a polymerase chain reaction to amplify a selected axon of a cDNA sequence of Figure Z., the use of oligonucleotide primers from intron portions near the 5~ and 3' boundaries of the selected axon of Figure 18.
BRTBF DBBCRIP°$°aoN of B'~~' lpR1',WIN~;B
Figure 1 is the nucleotide sequence of the CF gene and the amine acid sequence of the GFTR protein amino acid sequence with n indicating mutations at the 507 and 508 protein positions.
Figure 2 is a restriction map of the CF gene and the 2o schematic strategy used to chromosome walk and jump to the gene.
Figure 3 depicts the physical map of the region including and surrounding the CF gene generated by pulsed field gen electrophoresis. panels A, 8, C, and D shoWi hybridization data for the restriction enzymes Sal I, Xho I, Sfi T, and Nae I, respectively generated by representative genomic and cDNA probes which span the region. The deduced physical maps for each restriction enzyme is shown below each panel. A composite map of the entire MET-D7S8 interval is shown in panel E (J. M.
Rommens et al., Am. J. Hum. Genet. 45:932-947., 1990).
The open boxed segment indicates the portion cloned by chromosome walking and jumping, and the filled arrow indicates the gortion covered by the CF' transcript.
Figures 4A, 4B and 4C show the detection of conserved nucleotide sequences by cross-species hybridization.

wo ~m1o~34 1PCT/~CA97J00009 Figure 4D is a restriction map of overlapping segments of probes E4.3 and H1.6.
Figure 5 is an RNA blot hybridization analysis using genomic and cDNA probes. Hybridization to RNA of: A-fibroblast with cDNA probe ~-2; B-trachea (from unafflicted and CF patient individuals), pancreas, liver, HL6o cell line and brain with genomic probe CF16; C-T84 cell line with cDNA probe 10-1.
Figure 6 is the methylation status of the E4.3 1o cloned region at the 5~ end of the CF gene.
Figure ? is a restriction map of the CFTR cDNA
showing alignment of the cDNA to the genomic DNA
fragments.
Figure 8 is an RNA gel blot analysis depicting hybridization by a portion of the CFTR cDNA (alone 10-1) to a 6.5 kb mRNA transcript in various human tissues.
Figure 9 is a DNA blot hybridization analysis depicting hybridization by the CFTR,cDNA clones to genomic DNA digested with EcoRI and Hind III.
Figure 10 is a primer extension experiment characterizing the 5~ and 3~ ends of the CFTR cDNA.
Figure 12 is a hydropathy profile and shows predicted secondary structures of CFTR.
Figure Z2 is a dot matrix analysis of internal homologies in the predicted CFTR polypeptide.
Figure 13 is a schematic model of the predicted CFTR
protein.
Figure 14 is a schematic diagram of the restriction fragment length polymorphisms (RFLP~s) closely linked to the CF gene where the inverted triangle indicates the location of the F508 3 base pair deletion.
Figure 15 represents alignment of the most conserved segments of the extended NBFs of CFTR with comparable regions of other proteins.
Figure 16 is the DNA sequence around the F50~
deletion.

WO 91/107341 PCIf/CA91/00009 r .:
I~ ;.

Figure 17 is a representation of the nucleotide sequencing gel showing the DNA sequence at the F508 deletion.
Figure 18 is the nucleotide sequence of the portions of introns and complete axons of the genomic CF gene for 27 axons identified and numbered sequentially as 1 through 24 with additional ex~ns 6a, 6b, 14a, 14b and 17a, 17b of cDNA sequence of Figure l;
Figure 13 shows the results of amplification of l0 genomic DNA using intron oligonucleotides bounding axon 10e Figure 20 shows the separation by gel electrophoresis of the amplified genomic DNA products of a CF family; and Figure ~1 is a restriction mapping of cloned intron and exan portions of genomic DNA which introns and exans are identified in Figure 18.
~1~~AI~~D DE~CR~~'~IC1Y~ OF 'f81; kktEFERIt~D 1,~1~ODIl~k'NTg ,~,s. D~%F~Rt~'f~~I~Y~
In order to facilitate review of the various embodiments of the invention and an understanding of various elements and constituents used in,making the invention and using same, the following definitian of terms used in the invention description is as follows:
eF - cystic fibrosis CF carrier - a person in apparent health whose chromosomes contain a mutant CF gene that may be transmitted to that personls offspring.
CF patient - a person who carries a mutant CF gene on each chromosome, such that they exhibit the clinical symptoms of cystic fibrosis.
CF gene - the gene whale mutant forms are associated with the disease cystic fibrosis. This definition is understood to include the various sequence polymorphisms that exist, wherein nucleotide substitutions in the gene sequence do not affect. the essential function of the gene product. This term primarily relates to an isolated WO 9111074 pC f/CA91 /00009 ?~~3~~~~.
coding sequence, but can also include some or all of the flanking regulatory elements and/or introns.
Genomic CF gene - the CF gene which includes flanking regulatory elements and/or introns at baundaries of crone of the CF gene.
CF - PI - cystic fibrosis pancreatic insufficient, the major clinical subgroup of cystic fibrosis patients, characterized by insufficient pancreatic exocrine function.
CF - pS - cystic fibrosis pancreatic sufficient, a clinical subgroup of cystic fibrasis patients with sufficient pancreatic exocrine function for normal digestion of food.
CFTR ~ cystic fibrosis transmembrane conductance regulator protein, encoded by the CF gene. This t3efbr~ition includes the protein as isolated from human or animal sources, as produced by recombinant organisms, and as chemically or enzymatically synthesized. This definition is understood.ta include the various polymorphic forms of the protein wherein amino acid substitutions in the variable regions of the sequence does not affect the essential functioning of the protein, or its hydropathic profile or secondary or tertiary structure.
DNA - standard nomenclature is used to identify the bases.
Intronless DNA - a piece of DNA lacking internal non-coding segments, for example, cDNA.
IRP locus sequence - (protooncogene int-1 related), a gene located near the CF gene.
Mutant CFTR - a protein that is highly analagous to CFTR in terms of primary, secondary, and tertiary structure, but wherein a small number of amino acid substitutions and/or deletions and/or insertions result in impairment of its essential function, so that organisms whose epithelial sells express mutant CFTR

rather than CFTR demonstrate the symptoms of cystic fibrosis.
mCF - a mouse gene orthologous to the human CF gene 5 NBFs - nucleotide (ATP) binding folds ORF - open reading frame PCR - polymerase chain reaction Protein - standard single letter nomenclature is used to identify the amino acids 10 R-domain - a highly charged cytoplasmic domain of the CFTR protein RSV - Rous Sarcoma Virus SAP - surfactant protein RFLP - restriction .fragment length polymorphism 507 mutant CF gene - the; C'.F gene which includes a DNA base pair mutation 15 at the 506 or 507 protein position of the cDNA of the CF gene 507 mutant DNA sequence - equivalent meaning to the 507 mutant CF gene 507 mutant CFTR protein or mutant CFTR protein amino acid sequence, or mutant CFTR polypeptide - the mutant CFTR protein wherein an amino acid deletion occurs at the isoleucine 506 or 507 protein position of the CFTR.
Protein position means amino acid residue position.
2. ISOLATING THE CF GENE
Using chromosome walking, jumping, and cDNA hybridization, DNA
sequences encompassing > 500 kilobase pairs (kb) have been isolated from a region on the long arm of human chromosome 7 containing the cystic fibrosis (CF) gene.
This technique is disclosed in detail in the aforementioned United States Patent 5,776,677. For purposes of convenience in understanding and isolating the CF
gene and identifying other mutations, such as at the 85, 148, 1178, 455, 493, 507, 542, 549, 560, 563, 574, 1077 and 1092 amino acid residue positions, the technique is reiterated here. Several transcribed sequences and conserved segments have been identified in this region. One of these corresponds WO 91/10734 P(.'T/CA91/00009 to the Cc, gene and spans approximately 250 kb of genomic DNA. bverlapping complementary DNA (cDNA) clones have been isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the cystic S fibrosis gene. The nucleotide sequence of the isolated cDNA is shown in Figures 1 through 18. In each row of the respective sequences the lower row is a list by standard nomenclature of the nucleotide sequence. The upper row in each respective row of sequences is standard single letter nomenclature for the amino acid corresponding to the respective codon.
Accordingly, the isolation of the CF gene provided a cDNA molecule comprising a DNA sequence selected from the group consisting of:
(a) DNA sequences which correspond to the DNA
sequence of Figure 1 from amino acid residue position 1 to po~:2.tion 1480;
(b) DNA sequences encoding normal CFTR polypeptide having the sequence according to Figure 1 for amino acid 2o residue positions from 1 to 1480;
(c) DNA sequences which correspond to a fragment of the sequence of Figure 1 including at least 16 sequential nucleotides between amino acid residue positions 1 and 1480;
(d) DNA sequences which comprise at least 16 nucleotides and encode a fraganent of the amino acid sequence of Figure 1; and (e) DNA sequences encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of Figure 1 between amino acid residue positions 1 and 1480.
According to this invention, the isolation of other mutations in the CF gene alsa provides a cDNA molecule comprising a DNA sequence selected from the group consisting of:
a) DNA sequences which correspond to the DNA
sequence encodinglmutant CFTR polypeptide characterized by cystic fibrosis-associated activity inhuman dvo 9~rio~~a PcrrcA9~roooo9 17 v. ~
rd~ ' 4.3':'t ~.f epithelial cells, or the DNA sequence of Figure 1 for the amino acid residue positions 1 to 1480 yet further characterized by a base pair mutation which results in the deletion of or a change for an amino acid at residue positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092;
b) DNA sequences which correspond to fragments of the mutant portion of the sequence of paragraph a) and which include at least sixteen nucleotides;
c) DNA sequences which comprise at least sixteen nucleotides and encode a fragment of the amino said sequence encoded for by the mutant portion of the DNA
sequence of paragraph a); and d) DNA sequences encoding an epitope encoded by at least 18 sequential nucleotides in the mutant poz°tion of the sequence of the DNA of paragraph a).
Transcripts of approximately 6,500 nucleotides in size are detectable in tissues affected in patients with CF. Hosed upon the isolated nucleotide sequence, the 2o predicted protein consists of two similar regions, each containing a first domain having properties consistent with membrane association and a second domain believed to be involved in ATP binding.
A 3 by deletion which results in the omission of a phenylalanine residue at the center of the first predicted nucleotide binding domain (amino acid position 508 of the CF gene product) was detected in CF patients.
This mutation in the normal DNA sequence of Figure 1 corresponds to approximately 70% of the mutations in 3o cystic fibrosis patients. Extended haplotype data based on DNA markers closely linked to the putative disease gene suggest that the remainder of the CF mutant gene pool consists of multiple, different mutations. This is now exemplified by this invention at, for example, the 506 ar 507 protein position. A small set of these latter mutant alleles (approximately 8%) may confer residual W~ 91/1073a 1'CT/CA91/00009 pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient.
2.1 CH120MOBO1~IE W~,7GkCyNB ND ~'~MMpING
Large amounts of the DNA surrounding the D7S122 and D75340 linkage regions of Rommens et al supra were searched for candidate gene sequences. In addition to conventional chromosome walking methods, chromosome jumping techniques were employed to accelerate the search process. From each jump endpoint a new bidirectional l0 walk could be initiated. Sequential walks halted by "unclonable" regians often encountered in the mammalian genome could be circumvented by chromosome jumping.
The chramosome jumping library used has been described previously [Collins et al, S~,ience_ 235, 1046 (1987);W anuzzi et al, Am J ~,ium Genet 44, 695 (1989)]. The original library was prepared from a preparative pulsed field gel, and was intended to contain partial EcoRl fragments of 70 - 130 kb; subsequent experience with this library indicates that smaller fragments were also represented, and jumpsizes of 25 -110 kb have been found. The library was plated on sup host 1!1C1061 and screened by standard techniques, [Maniatis et al]. Positive clones~were subcloned into pBRo23Ava and the beginning and end of the jump identified by EcoRi and Ava 1 digestion, as described in COllinS, Genome anallrsiso ~, taraCtiCa~ ar~nrnanh ~~~,, London, 1988), pp. 73-94) For each clone, a fragment from the end of the jump was checked to confirm its location on chromosome 7. The contiguous chromosome region covered by chromosome walking and jumping was abaut 250 kb. Direction of the jumps was biased by careful choice of probes, as described by Collins et al and Ianuzzi et al, supra. The entire region cloned, including the sequences isolated with the use of the CF
gene cDNA, is approximately 500 kb.
The schematic representation of the chromosome walking and jumping strategy is illustrated in Figure 2.

iV0 91 / 10734 PCT/Cf191 /00009 '' ~ '~ '~ ,i;~ ') CF gene axons are indicated by Roman numerals in this Fire. Horizontal lines above the map indicate walk steps whereas the arcs above the map indicate jump steps.
The Figure proceeds from left to right in each of six tiers with the direction of ends toward 7cen and 7qter as indicated. The restriction map for the enzymes EcoRI, HindIII, and BamHT is shown above the solid line, spanning the entire cloned region. Restriction sites indicated with arrows rather than vertical lines indicate l0 sites which have not been unequivocally positioned.
Additional restriction sites for other enzymes are shown below the line. Gaps .in the cloned region are indicated bY ~~. These occur only in the portion detected by eDNA
clones of the CF transcript. These gaps are unlikely to be large based on pulsed field mapping of the region.
The walking clones, as indicated by horizontal~arrows above the map, have the direction of the straw indicating the walking progress obtained with each clone. Cosmid clones begin with the letter c; all other clones are phage. Cosmid CF26 proved to be a chimera; the dashed portion is derived from a different genomic fragment on another chromosome. Roman numerals I through XXIV
indicate the location of axons of the CF gene. The horizontal boxes shown above the line are probes used during the experiments. Three of the probes'represent independent subcloning of fragments previously identified to detect polymorphisms in this region: H2.3A corresponds to probe XV2C (X. EstiVill et al, at , 326: 840 (1987)), probe E1 corresponds to KM19 (Estivill, supra), and probe E4.1 corresponds to Mp6d.9 (X. Estivill et al.
Am. J. Hum Ggnet 44 ,704 (1989)), G~2 is a subfragment of E6 which detects a transcribed sequence. 8161, 8159, and 8160 are synthetic oligonucleotides constructed from parts of the IRP locus sequence [B. J. Wainwright et al, O J., 7: 1743 (1988)], indicating the location of this transcript on the genomic map.

As the two independently isolated DNA markers, D7S 122 (pH131) and D7S340 (TM58), were only approximately 10 kb apart (Figure 2), the walks and 5 jumps were essentially initiated from a single point. The direction of walking and jumping with respect to MET and D7S8 was then established with the crossing of several rare-cutting restriction endonuclease recognition sites (such as those for Xho I, Nru I and Not I, see Figure 2) and with reference to the long range physical map of A.M. Poustka, et al, Genomics 2, 337 (1988); M.L. Drumm et al. Genomics 2, 346 10 (1988). The pulsed field mapping data also revealed that the Not I site identified by the inventors of the present invention (see Figure 2, position 113 kb) corresponded to the one previously found associated with the IRP locus (Estivill et al 1987, supra).
Since subsequent genetic studies showed that CF was most likely located between IRP and D7S8 [M. Farrall et al, Arn. J. Hum. Genet. 43, 471 (1988), B.S. Kerem et al.
15 Am. J. Hum. Genet. 44, 827 (1989)], the walking and jumping effort was continued exclusively towards cloning of this interval. It is appreciated, however, that other coding regions, as identified in Figure 2, for example, G-2, CF14 and CF16, were located and extensively investigated. Such extensive investigations of these other regions revealed that they were not the CF gene based on genetic data and sequence 20 analysis. Given the lack of knowledge of the location of the CF gene and its characteristics, the extensive and time consuming examination of the nearby presumptive coding regions did not advance the direction of search for the CF
gene.
However, these investigations were necessary in order to rule out the possibility of the CF gene being in those regions.
Three regions in the 280 kb segment were found not to be readily recoverable in the amplified genomic libraries initially used. These less clonable regions 21 ~~~~~~~'~'t were 1~cated near the DNA segments H2.3A and ?C.6, and just~beyond cosmid cW44, at,positions 75-100 kb, 205-225 kb, and z75-285 kb in Figure 2, respectively. The recombinant clones near H2.3A were found to be very unstable with dramatic rearrangements after only a few passages of bacterial culture. To fill in the resulting gaps, primary walking libraries were constructed using special host-vector systems which have been reported to allow propagation of unstable sequences [A. R. Wyman, L.
B. Wolfe, D. Botstein, _proc. Nat Acad Sci ~r a ~ g2~
2880 (1985); K. F. Wertman, A. R. Wyman, D. Botstein, Gene 49, 253 (1986); A. R. Wyman, K. F. Wertman, D.
Barker, c. Helms, W. H. Petri, Gene, 49, 263 (1966)), Although the region near cosmid cW44 remains to be recovered, the region near X.6 was successfully rescued with these libraries.
COZdBTRDC'~T~At OF l3gNO~trr r °~~~~or~a Genomic libraries were constructed after procedures described in Manatis, et al, Nalecu~ar Clon~tng~
Laby g4~ (Cold Spring Harbor Laboratory, Cold Spring Harbor, New Yark 1982) and are listed in Table i.
This includes eight phage libraries, one of which was provided by T. Maniatis [Fritsch et al, Ce , 19:959 (1980)]; the ~°est were constructed as part of this work according to procedures described in l~aniatis et al, su~~a. Four phage libraries were cloned in aDASH
(commercially available from Stratagene) and three in aF~X (commercially available from Stratagene), with vector arms provided by the manufacturer. One aDASH
library was constructed from Sau 3A-partially digested DNA from a human-hamster hybrid containing human chromosome 7 (4AF/102/K015) [Rommens et al Am. ~' Hum a et 43, 4 (1988)], and other libraries from partial Sau3A, total BamHI, or total EcoRI digestion of human peripheral blood or lymphoblastoid DNA. To avoid loss of unstable sequences, five of the phage libraries were propagated on the recombination-deficient hosts DH1316 (recD-), CES 200 (recBC-;) [Wyman et al, supra , Wertman et al supra, Wyman et al supra]; or TAP90 [Patterson et al Nucleic Acids Res. 15:6298 (1987)]. Three cosmid libraries were then constructed. In one the vector pCV 108 [Lau et al Proc.
Natl.
Acad. Sci USA 80:5225 (1983)] was used to clone partially digested (Sau 3A) DNA
from 4AF/102/KO15 [Rommens et al Am.J. Hum. Genet. 43:4 (1988)]. A second cosmid library was prepared by cloning partially digested (Mbo I) human lymphoblastoid DNA into the vector pWE-IL2R, prepared by inserting the RSV
(Rous Sarcoma Virus) promoter-driven cDNA for the interleukin-2 receptor a-chain (supplied by M. Fordis and B. Howard) in place of the neo-resistance gene of pWElS
[Wahi et al Proc. Natl. Acad. Sci. USA 84:2160 (1987)]. An additional partial Mbo I
cosmid library was prepared in the vector pWE-1L2-Sal, created by inserting a Sal I
linker into the Bam HI cloning site of pWE-EL2R. This allows the use of the partial fill-in technique to ligate Sal I and Mbo I ends, preventing tandem insertions [Zabarovsky --et al Gene 42:19 (1986)]. Cosmid libraries were propagated in E.
coli host strains DH1 or 490A [M. Steinmetz, A. Winoto, K. Minard, L. Hood, Cell 28, WO 91110734 Pt.°TlCA91/00009 C3EP1~~i:~t~ x,lrB CBS

Vectr.~r Source of human DNA ~,gst ~om plexitxRef ~ Charon lIaeII/Alul-partially LE392 1 106 Lawn x 4A digested total human (amplified) et al liver DNA 1980 pCV108 Sau3a-gartially digested DK1 3 106 x DNA from 4AF/K015 (amplified) adash Sau3A-partially digested LE392 1 106 x DNA from 4AF/K015 (amplified) adash Sau3A-partially digested DB13161.5 x total human peripheral blood DNA

adash BamFiI-digested total DB1316 1.5 x human peripheral blood DNA

adash EcoRI-gartially digested DB1316S 10~
x total human peripheral blood DNA

aFIX Mbol-partially digested LE392 1.5 x 10g human lymphvblastoid DNA

3o aFlx Mbol-partially digested cE2oo 1.2 x lob human lymphoblastoid DNA

aFIX MboI-partially digested TAp90 1.3 x human lymphoblastoid DNA

pWE-IL2R P3boI-partially digested 490A 5 lOs x human lymphoblastoid DNA

pWE-IL2R- ~lbol-partially digested 490A 1.2 x Sal human.lymphoblastoid DNA

ACh3A EcoRI-partially digested MC10613 lOg x collins nlac (24-110 kb) et al (nu human lymphoblastoid DNA
mping) supra a d Iannuzzi et al supra w~ 91/10734 IPCT/CA91/00009 Three of the phage libraries were propagated and amplified in ~. co ' bacterial strain LE392. Four subsequent libraries were plated on the recombination-deficient hosts DB1316 (recD'~ or CES200 (rec BC's [Wyman 1985, su ra; Wertman 1986, su ; and Wyman 1986, sera]
or in one case TAP90 [T. A. Patterson and NI. Dean, Nucleic Acids Research 15, 6298 (1987)].
Single copy DNA segments (free of repetitive elements) near the ends of each phage or cosmid insert were purified and used as probes for library screening to isolate overlapping DNA fragments by standard procedures.
(Maniatis, et al, sutra).
1-2 x 106 phage clones were plated on 25-30 150 mm petri dishes with the appropriate indicator bacterial host and incubated at 37°C for 10-16 hr. Duplicate "lifts" were prepared for each plate with nitrocellulose ~;~:s nylon membranes, prehybridized and hybridized under conditions described [ltommens et al, 1988, supra].
Probes were labelled with 32P to a specific activity of >5 x 10a cpm/~cg using the random priming procedure (A. P.
Feinberg and ~. Vogelstein, glp,a~l. l~iochem. 132, 6 (1983)]. The cosmid library was spread on ampicillin-containing plates and screened in a similar manner.
DNA probes which gave high background signals could often be used more successfully by preannealing the boiled probe with 250 ~.g/ml sheared denatured placental DNA for 60 minutes prior to adding the probe to the hybridization bag.
For each walk step, the identity of the cloned DNA
fragment was determined by hybridization with a somatic cell hybrid panel to confirm its chromosomal location, and by restriction mapping and Southern blot analysis to confirm its colinearity with the genome.
The total combined cloned region of the genomic DNA
sequences isolated and the overlapping cDNA clones, extended >500 kb. To ensure that the DNA segments isolated by the chromosome walking and jumping procedures dV0 91!10734 PCT/CA91/00009 ~5 were colinear with the genomic sequence, each segment was examined bys (a) hybridization analysis with human-rodent somatic hybrid cell lines to confirm chromosome 7 localization, (b) pulsed field gel electrophoresis, and (c) comparison of the restriction map of the cloned DNA to that of the genomic DNA.
Accordingly, single copy human DNA sequences were isolated from each recombinant phage and cosmid clone and used as probes in each of these hybridization analyses as performed by the procedure of Maniatis, et al supra>
While the majority of phage and cosmid isolates represented correct walk and jump clones, a few resulted from cloning artifacts or cross-hybridizing sequences fram other regions in the human genome, or from the hamster genome in cases where the libraries were derived from a human-hamster hybrid cell line. Confirmation of correct localizatian was particularly important for clones isolated by chromosome jumping. Many jump clones were considered and resulted in non-conclusive information leading the direction of investigation away from the gene.
~s.~, CDNFIRM14,R~'~oTJ p~ R~gt~ s~~~~aTr~Tn~ MAP
s ,~. ya Further confirmation of the overall physical map of the overlapping clones was obtained by long rmnge restriction mapping analysis with the use ~f pulsed field gel electrophoresis (J. M. Rommens, et al. Am. J. ~Ium G net in press, A. M. Poustka et al, 1988, supra M.L.
Drumm et al, 1988 su ra).
Figures 3A to 3E illustrates the findings of the long range restriction mapping study, where a schematic representation of the region is given in Panel E. DNA
from the human-hamster cell line 4AF/102/K015 was digested t~ith the enzymes (A) Sal.I, (H) Xho I, (C) Sfi I
and (D) Nae I, separated by pulsed field gel electrophoresis, and transferred to zetaprobe~' (BioRad).
For each enzyme a single blot was sequentially hybridized with the probes indicated below each of the panels of Figure A to D, with stripping of the blot between hybridizations. The symbols for each enzyme of Figure 3E are:
A, Nae I; B, Bss HII; F. Sfi I; L, Sal I; M, Mlu I; N, Not I; R, Nru I; and X, Xho 1. C
corresponds to the compression zone region of the gel. DNA preparations, restnction digestion, and crossed field gel electrophoresis methods have been described ~
The gels in Figure 3 were run in 0.5X TBE at 7 volts/cm for 20 hours with switching linearly ramped from 10-40 seconds for (A), (B), and (C), and at 8 volts/cm for 20 hours with switching ramped linearly from 50-150 seconds for (D). Schematic interpretations of the hybridization pattern are given below each panel.
Fragment lengths are in kilobases and were sized by comparison to oligomerized bacteriophage 7~DNA and Saccharomyces cerevisiae chromosomes.
H4.0, J44, EG1.4 are genomic probes generated from the walking and jumping experiments (see Figure 2). J30 has been isolated by four consecutive jumps from D7S8 (Collins et al, 1987, supra; Ianuzzi et al, 1989, supra; M. Dean, et al, submitted for publication). 10-l, B.75, and CE1.5/1.0 are cDNA probes which cover different regions of the CF transcript: 10-1 contains exons I -VI, B.75 contains exons V
- XII, and CE1.5/1.0 contains exons XII - XXIV. Shown in Figure 3E is a composite map of the entire MET - D7S8 interval. The open boxed region indicates the segment cloned by walking and jumping, and the closed arrow portion indicates the region covered by the CF transcript. The CpG-rich region associated with the D7S23 locus (Estivill et al, 1987, supra) is at the Not I site shown in parentheses. This and other sites shown in parentheses or square brackets do not cut in 4AF/102/K015, but have __ . . , . , , ,_,,__~ __",.____ 27 ~~v~~~c'3~'t.a d.
..~ o xn~rrrx~~cA~rTorr ~~ cF
used on the findings of long range restriction mapping detailed above it was detenained that the entire CF gene is contained on a 38o kb Sal I fragment.
Alignment of the restriction sites derived from pulsed field gel analysis to those identified in the partially overlapping genomic DNA clones revealed that the size of the CF gene was approximately 250 kb.
The mast informative restriction enzyme that served to align the map of the cloned DNA fragments and the long range restriction map was Xho I; all of the 9 Xho 1 sites identified with the recombinant DNA clones appeared to be susceptible to at least partial cleavage in genomic DNA
(compare :asps in Figures Z and 2). Furthermore, hybridization analysis with probes derived frown the 3~
en~? of the CF gene identified 2 Sfil sites and confirmed the position of an anticipated Nae I site.
These findings further supported the canclusion that the DNA, segments isolated by the chromosome walking arid jumping procedures were colinear with the genuine sequence.
CRxTRR%1~1 F'oR '~i~RN'~,'TIa''r'G~Trn~
A positive result based on one or more of the following criteria suggested that a cloned DNA segment may contain candidate gene sequences:
(a) detection of cross-hybridizing sequences in other species (as many genes show evolutionary conservation), (b) identification of CpG islands, which often mark the 5~, end of vertebrate genes [A. P. bird, Natu,~g, 32Z~
209 (1986); M. Gardiner-Garden and M. Frommer, J~o~, Biol. 196, 261 (1987)], (c) examination of possible mRNA transcripts in tissues affected in CF patients, (d) isolation of corresponding cDNA sequences, (e) identification.of open reading frames by direct sequencing of cloned DNA segments.

WO 9]/10734 P('('/Cp9]/00009 -~;~c~~.

Cross-species hybridization showed strong sequence conservation between human and bovine DNA when CF'14, E4.3 and H1.6 were used as probes, the results of which are shown in Figures 4A, 4B and 4C.
Human, bovine, mouse, hamster, and chicken genomic DNAs were digested with Eco RI (R), Hind III (H), and Pst I (P), electrophoresed, and blotted to Zetabind~' (BioRad). The hybridization procedures of Rommens et al, 1988, su ra, were used with the most stringent wash at 55°C, 0.2X SSC, and 0.1% SDS. The probes used for hybridization, in Figure 4, included: (A) entire cosmid Cfl4, (B) E4.3, (C) H1.6. In the schematic of Figure (D), the shaded region indicates the area of cross-species conservation.
The fact that different subsets of bands were detected in bovine DNA with these two overlapping DNA
segments (H1.6 and E4.3) suggested that the conserved sequences were located at the boundaries of the overlapped region (Figure 4(D)). ~nThen these DNA segments were used to detect RNAwtranscripts from a variety of tissues, no hybridization signal was detected. In an attempt to understand the cross-hybridizing region and to identify possible open reading frames, the DNA sequences of the entire H1.6 and part of the E4.3 fragment were determined: The results showed that, except for a long stretch of CG-rich sequence containing the recognition sites for two restriction enzymes (Bss HI,I and Sac II), often found associated with undermethylated CpG islands, there were only short open reading frames which could not easily explain the strong cross-species hybridization signals.
To examine the methylation status of this highly CpG-rich region revealed by sequencing, genomic DNA
samples prepared from fibroblasts and lymphoblasts were digested with the restriction enzymes Hpa II and Msp I
and analyzed by gel blot hybridization. The enzyme Hpa IT cuts the DNA sequence 5~-CCGG-3' only when the second cytosine is unmethylated, whereas Msp I cuts this sequence regardless of the state of methylation. Small DNA fragments were generated by both enzymes, indicating that this CpG-rich region is indeed undermethylated in genomic DNA. The gel-blot hybridization with the E4.3 segment (Figure 6) reveals very small hybridizing fragments with both enzymes, indicating the presence of a hypomethylated CpG
island.
The above results strongly suggest the presence of a coding region at this locus. Two DNA segments (E4.3 and H1.6) which detected cross-species hybridization signals from this area were used as probes to screen cDNA
libraries made from several tissues and cell types.
cDNA libraries from cultured epithelial cells were prepared as follows. Sweat gland cells derived from a non-C',F individual and from a CF patient were grown to first passage as described [G. Collie et al, In Vitro Cell. Dev. Biol. 21, 592,1985].
The presence of outwardly rectifying channels was confirmed in these cells (J.A.
Tabcharani, T.J. Jensen, J.R. Riordan, J.W. Hanrahan, J. Memb. Biol., in press) but the CF cells were insensitive to activation by cyclic AMP (T.J. Jensen, J.W.
Hanrahan, J.A. Tabcharani, H. Buchwald and J.R. Riordan, Pediatric Pulmonolo~y, Supplement 2, 100, 1988). RNA was isolated from them by the method of J.M.
Chirgwin et al (Biochemistry 18, 5294, 1979). Poly A+RNA was selected (H. Aviv and P. Leder, Proc. Natl. Acad. Sci. USA 69, 1408, 1972) and used as template for the synthesis of cDNA with oligo (dT) 12-18 as a primer. The second strand was synthesized according to Gubler and Hoffman (Gene 25, 263, 1983). This was methylated with Eco RI methylase and ends were made flush with T4 DNA
polymerase. Phosphorylated Edo RI linkers were ligated to the cDNA and restricted with Eco RI. Removal of excess linkers and partial size fractionation was achieved by BiogelTM A-50 chromatography. The cDNAs were then ligated into the Edo RI
site of the commercially Wl7 91/10734 PCTlCA91/00009 available lamdba zAP. Recombinant were packaged and propagated ~in ~ cozy HH4. Portions of the packaging mixes.were amplified and the remainder retained for screening prior to amplification. The same procedures were used to construct a library from RNA isolated from preconfluent cultures of the T-84 colonic carcinoma cell line (Dharmsathaphorn, K. et al. Am. ,T. Phvs~ni. 246, 6204, 1984). The numbers of independent recombinant in the three libraries were: 2 x 106 for the non-CF sweat l0 gland cells, 4..5 x 106 for the CF sweat gland cells and 3.2 x 106 from T-84 cells. These phages were plated at 50,000 per 15 cm plate and plaque lifts made using nylon membranes (Biodyne) and probed with DNA fragments labelled with 'aP using DNA polymerise I and a random mixture of oligonucleotides as primer. Hybridization conditions were according to G.M. Wahl and S.L. Herger (Meth Enzymol. 152,415, 1987). Bluescript'~ plasmids were rescued from plaque purified clones by excision with M13 helper phage. The lung and pancreas libraries were purchased from Clontech Lab Inc. with reported sizes of 1.4 x 106 and 1.7 x 106 independent clones.
After screening 7 different libraries each containing 1 x lOs - 5 x 106 independent clones, 1 single clone (identified as l0-1) was isolated with H1.6 Eram a cDNA library made from the cultured sweat gland epithelial cells of an unaffected (non~CF) individual.
DNA sequencing analysis showed that probe 10-1 contained an insert of 920 by in size and one potential, long open reading frame (oRF). Since one end of the 3o sequence shared perfect sequence identity with H1.6, it was concluded that the cDNA clone was probably derived from this region. The DNA sequence in common was, however, only 113 by long (see Figures 1 and 7). As detailed below, this sequence in fact corresponded to the 5~-most exon of the putative CF gene. The short sequence overlap thus explained,the weak hybridization signals in library screening and inability tee detect transcripts in W~ 91/1fl734 Pt.'T/CA91/flfl009 r 31. ~.r~ s~e~~~~!i RNA gel-blot analysis. In addition, the orientation of the transcription unit was tentatively established on the basis of alignment of the genomic DNA sequence with the presumptive ORF of 10-1.
since the corresponding transcript was estimated to be approximately 6500 nucleotides in length by RNA gel~-blot hybridization experiments, further cDNA library screening was required in order to clone the remainder of the coding region. As a result of several successive to screenings with eDNA libraries generated from the colonic carcinoma cell line T84, normal and GF sweat gland cells, pancreas and adult lungs, 18 additional clones were isalated (Figure 7, as subsequently discussed in greater detail). DNA sequence analysis revealed that none of these cDNA clones corresponded to the length of the observed transcript, but it was possible to derive a censensus sequence based on overlapping regions.
Additional cDNA clones corresponding to the 5' and 3' ends of the transcript were derived from 5' and 3' primer-extension experiments. Together, these clones span a total of about 6.1 Icb and contain an ORF capable of encoding a polypeptide of 1480 amino acid residues (Figure 1).
It was unusual to observe that most of the cDNA
atones isolated here contained sequence insertions at various locations of the restriction map of Figure 7.
The map details the genomic structure of the CF gene.
Exon/intrc~n boundaries are given where all cDNA clones isolated are schematically represented on the upper half of the figure. Many of these extra sequences clearly corresponded to intron regions reversely transcribed during the construction of the cDNA, as revealed upon alignment with genomic DNA sequences.
Since the number of recombinant cDNA clones for the CF gene detected in the library screening was much less than would have been expected from the abundance of transcript estimated from RNA hybridization experiments, w0 91/10734 P(.°T/CA91/00009 it seemed probable that the clones that contained aberrant structures were preferentially retained while the proper clones were lost during propagation.
Consistent with this interpretation, poor growth was observed for the majority of the recombinant clones isolated in this study, regardless of the vector used.
The procedures used to obtain the 5° and 3° ends of the cDNA were similar to those described (M. Frohman et al, Proc. Nat. Aca~Sci, tTSA, 85, 8998-9002, 1988). Fox the 5r end clones, total pancreas and T84 poly A -~ RNA
samples were reverse transcribed using a primer, (10b), which is specific to exon 2 similarly as has been described for the primer extension reaction except that radioactive tracer was included in the reaction. The fractions collected from an agarase bead column of the first strand synthesis were assayed by polymerase chain reac~Lion (PCR) of eluted fractions. The oligonucleotides used were within the 10-1 sequence (245 nucleotides apart) just 5° of the extension primer. The earliest fractions yielding PCR product were pooled and concentrated by evaporation and subsequently tailed with terminal deoxynucleotidyl transferase (BRL Labs.) and dATP as recommended by the supplier (BRL Labs). A second strand synthesis was then carried out with Taq Polymerase (Cetus, AmpliTaq~') using an oligonucleotide containing a tailed linker.sequence 5°CGGAATTCTCGAGATC(T)123°.
amplification by an anchored (PCR) experiment using the linker. sequence and a primer just internal to the extension primer which possessed the Eco RI restriction site at its 5° end was then carried out. Following restriction with the enzymes Eco RI and Bgl II and agarose gel purification size selected products were cloned into the plasmid Bluescript KS available from Stratagene by standard procedures (Maniatis et al, supra). Essentially all of the recovered clones contained inserts of less than 350 nucleotides. To abtain the 3° end clones, first strand cDNA was prepared W~ 91/1073q QCT/CA91/00009 33 a ~~v~~.., ~;
with reverse transcription of 2 ag T04 poly A ~- RNA using the tailed linker oligonucleotide previously described with conditions similar to those of the primer extension.
Amplification by PCR was then carried c;ut with the linker oligonucleotide and three different oligonucleotides corresponding to known sequences of clone T16-4.5. A
preparative scale reaction (2 x 100 u1) was carried out with one of these oligonucleotides with the sequence 5'ATGAAGTCCAAGGATTTAG3'.
This oligonucleotide is approximately 70 nucleotides upstream of a Hind III site within the known sequence of T16-4.5. Restriction of the PCR product with Hind III
and Xho 1 was followed by agarose gel purification to size select a band at 1.0-1.4 kb. This product was then cloned into the plasmid Hluescript KS available from stratagene. Approximately 20~ of the obtained clones hybridized to the 3' end portion of T16-4.5. 10/10 of plasmids isolated from these clones had identical restriction maps with insert sizes of approx. 1.2 kl~.
All of the PCR reactions were carried out for 30 cycles in buffer suggested by an enzyme supplier.
An extension primer positioned 157 nt from the 5'end of 10-1 clone was used to identify the start point of the putative CF transcript. The primer was end labelled with -~(32P]ATP at 5000 Curies/mole and T4 polynucleotide kinase and purified by spun column gel filtration. The radiolab~led primer was then annealed with 4-5 ug poly A
-~ RNA prepared from T-84 colonic carcinoma cells in 2X
reverse transcriptase buffer for 2 hrs. at 60'C.
3o Following dilution and addition of AMV reverse transcriptase (Life Sciences, Inc.y incubation at 41'C
proceeded for 1 hour. The sample was then adjusted to 0.4M Na~H and 20 mM EDTA, and finally neutralized, with NH~OAc, pH 4.6, phenol extracted, ethanol precipitated, redissolved in buffer with formamide, and analyzed on a polyacrylamide sequencing gel. Details of these methods wo ~mo73a racricA~rioooo9 ~~ ii 34 have been described (Meth. EnzVmol. 152, 1987, Ed. S.L.
Bexger, A.R. ICimmel, Academic Press, N.Y.).
Results of the primer extension experiment using an extension oligonucleo~:ide primer starting 157 nucleotides from the 5~ end of 10-1 is shown in Panel A of Figure 10.
End labelled X174 bacteriophage digested with Hae III
(BRL Labs) is used as size marker. Two major products are observed at 216 and 100 nucleotides. The sequence corresponding to 10o nucleotides in 10-1 corresponds to a ZO very GC rich sequence (11/12] suggesting that this could be a reverse,transcriptase pause site. The 5~ anchored PCR results are shown in panel B of Figure 10. The 1.4%
agarose gel shown on the left was blotted and transferred to Zetaprobe~° membrane (Bio-Rad Lab). DNA gel blot hybridization with radiolabeled l0-1 is shown on the right. The 5~ extension products are seen to vary in size from 170-280 nt with the major product at about 200 nucleotides. The PCR control lane shows a fragment of 145 nucleotides. Tt was obtained by using the test oligomers within the 10-1 sequence. The size markers shown correspond to sizes of 154, 220210, 298, 344, 394 nucleotides (lkb ladder purchased from BRL Lab).
The schematic shown below Panel B of Figure 10 outlines the procedure to obtain double stranded cDNA
used for the amplification and cloning to generate the clones PA3-5 and TB2-7 shown in Figure 7. The anchored PCR experiments to characterize the 3~end are shown in panel. C. As depicted in the schematic below Figure 10C, three primers whose relative position to each other were known were used for amplification with reversed transcribed T84 RNA as described. These products were separated on a 1% agarose gel and blotted onto nylon membrane as described above. DNA-blot hybridization with the 3~ portion of the T16-4.5 clone yielded bands of sizes that corresponded to the distance between the specific oligomer ~used.and the 3~end of the transcript.
These bands in lanes 1, 2a and 3 are shown schematically WO 91 / 1073d PCT/CA91 /00009 3 5 ~ i~ ~~ ~ ~ r'~ ~.a.
below Panel C in Figure 10. The band in lane 3 is weak as only 60 nucleotides of this segment overlaps with the probe used. Also indicated in the schematic and as shown in the lane 2b is the product generated by restriction of the anchored PCR product to facilitate cloning to generate the THZ-d clone shown in Figure 7, DNA-blot hybridization analysis of genomic DNA
digested with EcoRI and HindIII enzymes probed with portions of cDNAs spanning the entire transcript suggest that the gene contains at least 26 axons numbered as Roman numerals I through XXVI (see Fi.c~ure 9), These correspond to the numbers 1 through 26 shown in Figure 7.
The size of each band is given in kb.
In Figure 7, open boxes indicate approximate positions of the 24 axons which have been.identified by the isolation of X22 clones from the screening of cDNA
libraries and from anchored PCR experiments designed to clone the 5~ and 3~ ends. The lengths in kb of the Eco RI genomic fragments detected by each axon is also 2o indicated. The hatched boxes in Figure 7 indicate the presence of intron sequences and the stippled boxes indicate other sequences. Depicted in the lower left by the closed box is the relative position of the clone H1.6 used to detect the first cDNA clone 10-1 from among 106 phage of the nox~al sweat gland library. As shown in Figures 4(D) and 7, the genomic alone H1.6 partially overlaps with an EcoRI fragment of X1.3 kb. All of the cDNA clones shown were hybridized to genomic DNA and/or were fins restriction mapped. Examples of the restriction sites occurring within the eDNAs and in the corresponding gendmic fragments are indicated, With reference to Figure 9, the hybridization analysis includes probes; i.e., cDNA clones 10-1 for panel A, T16-1 (3o portion) for panel B, T16~4.5 (central portion) for panel C and T16-4.5 (3~ end portion) for panel D. In panel~A of,Figure 9, the cDNA probe 10-1 detects the genomic bands for axons I through VI. The 3' WO 91/10734 PCTlCA91/00009 portion of T16-5. generated by NruI restriction detects axons IV through XIIT as shown in Panel B. This probe partially overlaps with 10-1. Panels C and D, respectively, show genomic bands detected by the central and 3' end EcoRI fragments of the clone T16-4.5. Two EcoRI sites occur within the cDNA sequence and split axons XIII and XIX. As indicated by the axons in.
parentheses, two genomic EcoRI bands correspond to each of these exans. Cross hybridization to other genomic 1o fragments was observed. These bands, indicated by N, are not of chromosome 7 origin as they did not appear in human-hamster hybrids containing human chromosome 7. The faint band in panel D indicated by XI in brackets is believed to be caused by the cross-hybridization of sequences due to internal homology with the cDNA.
Since l0-~ detected a strong band on gel blot hybridization of RNA from the T-8~ colonic carcinoma cell line, this cDNA was used to screen the library constructed from that source. Fifteen positives were obtained fro~a which clones T6, T6/20, T11, T16-1 and T13-1 were purified and sequenced. Rescreening of the same library with a 0.75 kb Bam HI-Eco RI fragment from the 3f end of T16-1 yielded T16-4.5. A l.8kb EcoRI fragment from the 3' end of T16-4.5 yielded T8-B3 and Tl2a, the latter of which contained a polyadenylation signal and tail. Simultaneously a human lung cDNA library was screened; many clones were isolated including those shown here with the prefix 'GDL'. A pancreas library was also screened, yielding clone CDp,TS.
To obtain copies of this transcript from a CF
patient, a cDNA library from RNA of sweat gland epithelial cells from a patient was screened with the 0.75 kb Sam HI - Eco RI fragment from the 3' end of T16-1 and clones C16-1 and C1-1/5, which covered all but axon 1, were isolated. These two clones both exhibit a 3 by deletion in axon 10 which is not present in any other clone containing that axon. Several clones, including 'WO 91/1073A PCT/CA91/00009 37 ~~r~~'k~~~
CDLS28-1 from the lung library and T6J20 and T13-1 isolated from T84 were derived from partially processed transcripts. This was confirmed by genomic hybridization and by sequencing across the exon-intron boundaries for each clone. T11 also contained additional sequence at each end. T16-4.5 contained a small insertion near the boundary between axons l0 and 11 that did not correspond to intron sequence. Clones CDLS16A, lla and 13a from the lung library also contained extraneous sequences of unknown origin. The clone C16-1 also contained a short insertion corresponding to a portion of the 7-transposon of E. coli; this element was not detected in the other clones. The 5' clones PA3-5, generated from pancreas RNA
and-TB2-7 generated from T84 RNA using the anchored PCR
technique have identical sequences except for a single nucleotide difference in length at the 5' end as'shown in Figure 1. The 3' clone, THZ-4 obtained from T84 RNA
contains the 3' sequence of the transcript in concordance with the.genomic sequence of this region.
A combined sequence representing the presumptive coding region of the CF gene was generated from overlapping cDNA clones. Since most of the eDNA clones were apparently derived from unprocessed transcripts, further studies were performed to ensure the authenticity of the combined sequence. Each cDNA clone was first tested for localization to chromosome 7 by hybridization analysis with a human-hamster somatic cell hybrid containing a single human chromosome 7 and by pulsed field gel electrophoresis. Fine restriction enzyme mapping was also performed for each clone. While overlapping regions were clearly identifiable far most of the clones, many contained regions of unique restriction patterns:
To further characterize these cDNA clones, they were used as probes in gel hybridization experiments with EcoRI -or HindIIZ-digested human genomic DNA. As shown in Figure 9, five to six different restriction fragments wo 9a~ao~3a Pcricn9aiomoo~
~~~~.~~a~~ ~ 3s could be detected with the 10-1 cDNA and a similar number of'fragments with ather cDNA clones, suggesting the presence of multiple axons for the putative CF gene. The hybridization studies also identified those cDNA clones with unprocessed intron sequences as they showed preferential hybridization to a subset of genomic DNA
fragments. For the confirmed cDNA clones, their corresponding genomic DNA segments were isolated and the axons and exon/intron boundaries sequenced. As indicated in Figure 7, at least 27 exoris have been identified which includes split axons 6a, 6b, 14a, 14b and 17a, 17b.
Based on this information and the results of physical mapping experiments, the gene locus was estimated to span 250 kb on chromosome 7.
2.6 THE BEOaJEIZCE
Figure 1 shows the nucleotide sequence of the cloned cD.~JA encoding CFTR together with the deduced amino acid sequence. The first base position corresponds to the first nucleotide in the 5' extension clone PA3-5 which is one nucleotide longer than TB2-7. Arrows indicate position of transcription initiation site by primer extension analysis. Nucleotide 6129 is followed by a poly(dA) tract. Positions of axon junctions are indicated by vertical lines. Potential membrane-spanning segments were ascertained using the algorithm of Eisenberg et al J. Mol. Bio~ 179:125 (1984). Potential membrane-spanning segments as analyzed and shown in Figure 11 are enclosed in boxes of Figure 1. In Figure 11, the mean hydropathy index [Kyte and Doolittle, J.
Molec. Biol. 157: 105, (1982)] of 9 residue peptides is plotted against the amino acid number. The corresponding positions of features of secondary structure predicted according to Garnier et al, [J. Molec a3so~ 157, 1s5 (1982)] are indicated in the lower panel. Amino acids comprising putative ATP-binding folds are underlined in Figure 1. Possible sites of phosphorylation by protein kinases A (PKA) or C (PKC) are indicated by open and 3~ ~G~ ~~~~~t closed circles, respectively. The open triangle is over the 3bp (CTT) which are deleted in CF (see discussion below). The cDNA clones in Figure 1 were sequenced by the dideoxy chain termination method employing 35S
labelled nucleotides by the Dupont Genesis 2000' automatic DNA sequencer.
The combined cDNA sequence spans 6129 bass pairs excluding the poly(A) tail at the end of the 3' untranslated region and it contains an ORF capable of encoding a polypeptide of 1480 amin~ acids (Figure 1).
An ATG (AUG) triplet is present at the beginning of this ORF (base position 133-X135). Since the nucleotide sequence surrounding this colon (5'pAGACCCA-3') has the proposed features of the consensus sequence (CC) Z5 A/GCCAUGG(G) of an eukaryotic translation initiation site with a highly conserved A at the -3 position, it is highly probable that this AUG~corresponds to the first msthionine colon for the putative polypeptide.
To obtain the sequence corresponding to the 5' end of the transcript, a primer-extension experiment was performed, as described earlier. As shown in Figure 10A, a primer extension product of approximately 216 nucleotides could be observed suggesting that the 5' end of the transcript initiated approximately 60 nucleotides upstream of the end of cDNA clone 10-1. A modified polymerase chain reaction (anchored PCR) was then used to facilitate cloning of the 5'-end sequences (Figure 10b).
Two independent 5'-extension clones, one from pancreas and the ether from T84 RNA, were characterized by DNA
30. sequencing and were found to differ by only 1 base in length, indicating the most probable initiatian site for the transcript as shown in Figure 1.
Since most of the initial cDNA clones did not contain a polyA tail indicative of the end of a mRNA, anchored PCR was also applied to the 3' end of the transcript (Frohman et. al, 1988, s_.upra). Three 3'-extension oligonucleotides were made to the terminal wo 9li~o~3a PCfl~A91/00009 ~~,~'~~i~i: ; 40 portion of the cDNA clone T16-4.5. As shown in Figure lOc, 3 PCR products of different sizes were Obtained.
All were consistent with the interpretation that 'the end of the transcript was approximately 1.2 kb downstream of the HindIII site at nucleotide position 5027 (see Figure 1). The DNA sequence derived from representative clones was in agreement with that of the T84 cI3NA cline Tl2a (see Figure 1 and ~) and the sequence of the corresponding 2.3 kb EcoRI genomlc fragment.
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To visualize the transcript for the putative CF
gene, RNA gel blot hybridization experiments were performed with the 10-l cDNA as probe. The RNA
hybridization results are shown in Figure 8.
RNA samples were prepared from tissue samples obtained fram surgical pathology or at autopsy according to methods previously described (A. M. Kimmel, S.L.
Bergen eds. Meth. Fn'~~.no~. 152, 1987)._ Formaldehyde 2o gels were transferred onto nylon membranes (zetaprobe TM~
BioRad Lab): The membranes were then hybridized with DNA
probes labeled to high specific activity by the random priming method (A. P. Feinberg and B. Vogelstein, Anal, Bioi chum 132, 6, 1983) according to previously published procedures (J. Rommens et al, Am. J. Hum Gent 43, 645-663, 1988). Figure 8 shows hybridization by the cDNA
clone 10-1 to a 6.5kb transcript -in the tissues indicated. Total RNA (10 ~cg) of each tissue, and Poly A+
RNA (1 fag) of the T84 colonic carcinoma cell line were separated on a 1% formaldehyde gel, The positions of the 28S and 18S rRNA bands are indicated. Arrows indicate 'the position of transcripts. Sizing was established by comparison to standard RNA markers (BRL Labs). HL60 is a human promyelocytic leukemia cell line, and T84 is a human colon cancer cell line.
Analysis reveals a prominent band of approximately 6.5 kb in size in T84 cells. Similar, strong iY~ 91 / I 0734 PC°T/CA91 /00009 ~;
~~ 1 ~ ~ .~ e~ ~t cs ~:..
hybridization signals were also detected in pancreas and primary cultures of cells from nasal polyps, suggesting that the mature mRNA of the putative CF gene is approximately 6.5 kb. Minor hybridization signals, probably representing degradation products, were detected at the lower size ranges but they varied between different experiments. Identical results were obtained with other cBNA clones as probes. Based on the hybridization band intensity and comparison with those detected for other transcripts under identical experimental conditions, it was estimated that the putative CF transcripts constituted approximately 0.01 of total mRNA in X84 cells.
A number of other tissues were also surveyed by RNA
gel blot hybridization analysis in an attempt to correlate the expression pattern of the 10-I gene hnd the pathology of CF. As shown in Figure 8, transcripts, all of identical size, were found in lung, colon, sweat glands (cultured epithelial cellsj, placenta, liver, and parotid gland but the signal intensities in these tissues varied among different preparations and were generally weaker than that detected in the pancreas and nasal polyps. Tntensity varied among different preparations, for example, hybridization in kidney was not detected in the preparation shown in Figure 8, but can be discerned in subsequent repeated assays. No hybridization signals cauld be discerned in the brain or adrenal gland (Figure 8j, nor in skin fibroblast and lymphoblast cell lines.
Tn summary, expression of the CF gene appeared to occur in many of the tissues examined, with higher levels in those tissues severely affected in CF. While this epithelial tissue-specific expression pattern is in good agreement with the disease pathology, no significant difference has been detected in the amount or size of transcripts from CF and control tissues, consistent with the assumption that CF mutations are subtle changes at the nucleotide level.

CVO 91/10734 PCTlCA91100009 3.2 TFI~ 3'~~OR CF' ~IUTA.T,~~~1 Figure 16 shows the DNA sequence at the F508 deletion. On the left, the reverse complement of the sequence from base position 1649-1664 of the normal sequence (as derived from the cDNA clone T16). The nucleotide sequence is displayed as the output (in arbitrary fluorescence intensity units, y-axis) plotted against time (x-axis) for each of the 2 photomultiplier tubes' (P2~T~1 and ,~2) of a Dupont Genesis 2000TM DNA
l0 analysis system. The corresponding nucleotide sequence is shown underneath. On the right is the same region from a mutant sequence (as derived from the cDNA clone C16). Double-stranded plasmid DNA templates were prepared by the alkaline lysis procedure. Five ~Sg of plasmid DNA and 75 ng of oligonucleotide primer were used in each sequencing reaction according to the protocol recommended by Dupont except that the annealing was done at 45°C for 3o min and that the elongation/termination step was for l0 min at 42°C. The unincorporated fluorescent nucleatides were remaved by precipitation of the DNA sequencing reaction product with ethanol in the presence of 2.5 M ammonium acetate at pH 7.0 and rinsed one time with 70~ ethanol. The primer used for the T16-1 sequencing was a specific oligonucleotide 5'GTTGGCATGCTTTGATGACGCTTC3° spanning base position 1708 - 1731 and that far C16-1 was the universal primer SK far the Bluescript vectar (Stratagene).
Figure 17 also shows the DNA sequence araund the F508 deletion, as determined by manual sequencing. The normal sequence from base pasition 1726-1651 (from cDNA
T16-1) is shown beside the CF sequence (from cDNA C16-1).
The left panel shows the sequences from the coding strands obtained witty the B primer (5'GTTTTCCTGGATTATGCCTGGCAC3') and the right panel those from the opposite strand with the D primer (5'GTTGGCATGCTTTGATGACGCTTC3'). The brackets indicate the three nucleotides in the normal that are absent in CF

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(arrowheads). Sequencing was performed as described in F. Sanger, S. Nicklen, A. R. Coulsen, Proc. Nat. Acad.
sci. u. s. A. ?~: 5463 (i9??~.
The extensive genetic and physical mapping data have directed molecular cloning studies to focus on a sanall segment of DNA on chromosome 7. Because of the lack of chromosome deletions and rearrangements in CF and the lack of a well-developed functional assay for the CF gene product, the identification of the CF gene required a detailed characterization of the locus itself and comparison between the CF and normal (N) alleles.
Random, phenotypically normal, individuals could not be included as controls in the comparison due to the high frequency of symptomless carriers in the population. As a result, only parents of CF patients, each of whom by definition carries an N and a CF chromosome, were suitable for the analysis. Moreover, because of the strong allelic association observed between CF and some of the closely linked DNA markers, it was necessary to exclude the possibility that sequence differences detected between N and CF were polymorphisms associated with the disease locus.
3.3 IDENT~FI~~fJL"ION OF RFLPs AND FAMIh'X sT~TD~Es To determine the relationship of each of the DNA
segments isolated from the chromosome walking and jumping experiments to CF, restriction fragment length polymorphisms (RFLPs) were identified and used to study families where crossover events had previously been detected between CF and other flanking DNA markers. As shown in Figure 14, a total of 18 RFLPs were detected in the 500 kb region; 1? of them (from E6 to CE1.0) listed in Table 2; some of 'them correspond to markers previously reported.
Five of the RFLPs, namely 10-1X.6, T6/20, H1.3 and CE1.0, were identified with cDNA and genomic DNA probes derived from the putative CF gene. The RFLP data are presented in Table 2, with markers in the MET and D?S8 W~ 91 /10734 PCTlCA91 /00009 k as regions included for comparison. The physical distances between these markers as well as their relationship to the M>;T and D7S8 regions are shown in Figure 14.

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p7 w0 93/10734 PCT/CA9i/00009 P~IO~'~8 FOR 'f~IBTW 2 (a) The number of N and CF-PT (CF with pancreatic insufficiency) chromosomes were derived from the parents in the families used in linkage analysis 5 [Tsui et al, Cold Spring Harbor Sump Ouant Biol 51:325 (1986)].
(b) Standardized association (A), which is less influenced by the fluctuation of DNA marker allele Z~ distribution among the N chromosomes, is used here for the comparison Yule~s association coefficient A=(ad-bc)/(ad+bc), where a, b, c, and d are the number of N chromosomes with DNA marker allele l, CF
with 1, N with 2,. and CF with 2 respectively.
15 Relative risk can be calculated using the relatianship RR = (I~A)/(1-A) or its reverse.
Allelic association (*), calgulated according to A.
Chakravarti et al, Am. J Hum cP~e~ 36:1239, 20 (3.984) assuming the frequency of 0.02 for CF
chromosomes in the population is included for, comparison.
Because of the small number of recombinant families 25 available for the analysis; as was expected from the close distance between the markers studied and CF, and the possibility of misdiagnosis, alternative approaohes were neoessary in further fine mapping of the CF gene.
3.4 L ~h C A88oCIA2~~eat 3a Allelic association (linkage disequilibrium) has been detected for many closely linked DNA markers. While the utility of using allelic association for measuring genetic distance is uncertain, an overall correlation has been observed between CF and the flanking DNA markers. A
35 strong association with CF was noted for the closer DNA
markers, D7S23 and D7S122, whereas little or no WO 91 /10734 PCT/CA91I0000~
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association was detected for the more distant markers MET, D7S8 or D7S424 (see Figure 1).
As shown in Table 2, the degree of association between DNA markers and CF (as measured by the Yule's 5 association coefficient) increased from 0.35 for metes and 0.17 for J32 to 0.91 for 10-1X.6 (only CF-PI patient families were used in the analysis as they appeared to be genetically more homogeneous than CF-PS). The association coefficients appeared to be rather constant over the 300 kb from EG1.4 to H1.3; the fluctuation detected at several locations, most notably at H2.3A, E4.1 and T6/20, were probably due to the variation in the allelic distribution among the N chro2~osomes (see Table 2). These data are therefore consistent with the result from the study of recombinant families (see Figure 14).
A similar conclusion could also be made by inspection of the extended DNA marker haplotypes associated with the CF
chromosomes (see below). However, the strong allelic association detected over the large physical distance between EGl.4~and Fil.3 did not allow further refined mapping of the CF gene. Since J44 was the last genomic DNA clone isolated by chromosome walking and jumping before a eDNA clone was identified, the strong allelic association detected for the JG2E1-J44 interval prompted us to search for candidate gene sequences over this entire interval. It is of interest to note that the highest degree of allelic. association was, in fact, detected between CF and the 2 RFLPs detected by 10-1X.'6, a region near the major CF mutation.
Table 3 shows pairwise allelic association between DNA markers closely linked to CF. The average number of chromosomes used in these calculations was 75-80 and only chromosomes from CF-PI families were used in scaring CF
chromosomes. Similar results were obtained when Yule's standardized association (A) was used.

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wo 9W lo~3a PCT/CA91/000~9 53 ~~;~~'':z'x,' Strong allelic association was else detected among subgroups of RFLFs on bath the CF and N chromosomes. As shown in Table 3, the DNA markers that are physically close to each other generally appeared to have strong association with each other. For example, strong (in some cases almost complete) allelic association was detected between adjacent markers E6 and E7, between pH131 and W3D1.4~between the AccT and HaeTTT polymorphic sites detected by 10-1X.6 and amongst EG1.4, JG2E1, E2.6(E.9), E2.8 and E4.1. The two groups of distal markers in the MET and D?S8 region also showed some degree of linkage disequilibrium among themselves but they showed little association with markers from E6 to CE1.0, c~nsistent with the distant locations for MET and D7S8. On the other hand, the lack of association between DNA markers that are physically close may indicate the pres~:nca of recombination hot spots. Examples of these potential hot spots are the region between E'T and pH131, around H2.3A, between J44 and the regions covered by the pr~bes 10-1X.6 and T6/20 (see Figure 14). These regions, containing frequent recombination breakpoints, were useful in the subsequent analysis of extended haplotype data for the CF region.
3 a 5 ~iA~~!1'YPL ANA.LYBTB
Extended haplotypes based on 23 DNA markers were generated for the CF and N chr~mosomes in the collection of families prewiausly used for linkage analysis.
Assuming recombination between chromosomes of different haplotypes, it was possible to construct several lineages of the observed CF chromosomes and, also, to predict the location of the disease locus.
To obtain further information useful for understanding the nature of different CF mutations, the F508 deletion data were correlated with the extended DNA

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i~'O 91110734 FGf/CA91 /~~DOU9 TAELE 4 (continued) (a) The extended haplotype data are derived from the CF
families used in previous linkage studies (see footnote (a) of Table 3) with additional CF-PS families collected 5 subsequently (Kerem et a1, Am. J. GeDet. 44:827 (1989)).
The data are shown in groups (regions) to reduce space.
The regions are assigned primarily according to pairwise association data shown in Table 4 with regions 6-8 spanning the putative CF locus (the F508) deletion is l0 between regions 6 and 7). A dash (-) is shown at the region where the haplotype has not been deter~ainsd due to incomplete data or inability to establish phase.
Alternative haplotype assignments are also given where date are incomplete. Unclassified includes those 15 chromosomes with more than 3 unknown assignments. The hap~,o-~ype definitions for each of the 9 regions are:
Region 1- metD metD mates Banl 2o A = 1 1 1 B = 2 1 2 C g 1 1 2 D = 2 2 1 E = 1 2 25 F = 2 Z 1 G = 2 2 2 Region 2- E6 E7 pHI31 W3D1.~

30 Taal Taa A = 1 2 2 2 s = 2 Z

C = 1 2 1 1 35 D = 2 1 2 2 E = 2 ~ 2 2 1 F = 2 2 1 1 67.

G = 1 2 1 2 H = 1 1 2 2 Region 3- H2.3A

Taqz_ A = 1 B = 2 Region 4- EC1.4 EG1.4 JG2E1 ~,~n~~x Ba~.x ~Stz A = 1 1 2 B = 2 2 1 C = 2 2 2 D = 1 l 1 E = 1 2 1 ~0 Region 5- E2.6 E2.8 E4.1 NCA MspI

A a 2 1 2 B ~ 1 2 1 C = 2 2 2 Region 6- J44 10-1X.610-1X.6 3 t1 Xbal cc HaeIII

A = 1 2 1 B = 2 1 2 C = 1 1 2 D = 1 2 2 E = 2 2 2 F = 2 2 1 ii~0 91!30734 ~'Cd'/CA91100009 r ~,~~~~~ ~ 62 RegJ.oI1 7° T6~20 MS1~I
A = 1 B = 2 Region 8- H1.3 CE 1.0 co eI

to A = 2 1 B= 1 2 c= 1 1 D = 2 2 Region 9- J32 J3.11 J29 Sac NisnI PvuII

A = 1 1 1 B = 2 2 2 C = 2 1 2 D = 2 2 1 E = 2 1 1 (b) Plumber of chromosomes scored in each class:
CF-PI(F) = CF chromosomes from CF-PI patients with the F508 deletion;
CF-PS(F) = CF chromosomes from CF-PS patients with the F508 deletion;
CF-PI = Other CF chromosomes from CF-PI patients;
CF-PS = Other CF chromosomes from CF-PS patients;
N = Normal chromosomes derived from carrier parents dV~ 91 / 10734 PC'1!'/CA.91 /00009 63 ~:::~f ~ ~ ~) tJ i1 ~ t' It was apparent that most recombinations between haplotypes occurred between regions Z and 2 and between regions 8 and 9, again in good agreement with the relatively long physical distance between these regions.
Other, less frequent, breakpaints were noted between short distance intervals and they generally corresponded to the hot spots identified by pairwise allelic association studies as shown above. It is of interest to note that the F508 deletion associated almost exclusively 1C with Group I, the most frequent CF haplotype, supporting the position that this deletion constitutes the major mutation in CF. I4lore important, while the F508 deletion was detected in 89~ (62/70) of the CF chromosomes with the AA haplotype (corresponding to the two regions, 6 and 7) flanking the deletion, it was not was found in the 14 N chromosomes within the same group (~2 = 47.3, p <10'~).
The ~'S08 deletion was therefore not a sequence polymorphism associated with the core of the Group I
haplotype (see Table 5).
Together, the results of the oligonucleotide hybridization study and the haplotype analysis support the fact that the gene locus described here is the CF
gene and that the 3 by (F508) deletion is the most common mutation.in CF.
3.6 INTRONjEXON HOONDhRIER
The entire genomic CF gene includes all of the regulatory genetic information as well as intron genetic information which is splicad out in the expression of the CF gene. Portions of the introns at the intron/exon boundaries for the exons of the CF gene are very helpful in lacating mutations in the CF gene, as they permit PCR
analysis from genomic DNA. Genomic DNA can be obtained from any tissue including leukocytes from blood. Such intron information can be employed in PCR analysis for purposes of CF screening which will be discussed in more detail in a later section. As set out in Figure 18 with the headings "Exon 1 through Exon 24", there are portions 6V0 91/10734 ~'C1'/CA91100009 s4 of the bounding introns in particular those that flank the axons which are essential for PCR axon amplification.
Further assistance in interpreting the information of Figure 18 is provided in Figure 21. Genomic DNA
clones containing the coding region of the CFTR gene axe provided. As is apparent from Figure 21, there are considerable gaps between the clones of the axons which indicates the gaps in the intron portions between the axons of Figure 18. These gaps in the intron portions to are indicated by 'o.,,e~. In Figure 21, the clones were mapped using different restriction endonucleases (AccI,A;
AvaI,W; BamFiI,B; BgIII,G; BssIiI,Y; EcoRV,V; FspI,F;
HinCII,C; FIIndIII,H; Kpn,K; NCOI,.T; PstI,P; PVUII,U;
SmaI,M; SacI,S; SspI,E; StyI,T; XbaI,X; XhoI,O). Iri Figure 21, the axons are represented by boxed regions.
The ~apen boxes indicate non-coding portions of the axons, whereas closed boxes indicate Boding portions. The probable positions of the axons within the genomic DNA
are also indicated by their relevative positions. The arrows above the boxes mark the location of the oligonucleotides used as sequencing primers in the PCR
amplification of the genomic DNA. The numbers provided beneath the restriction map represent the size of the restriction fragments in kb.
Tn sequencing the intron portions, it has been determined that there are at least 27 axons instead of the previously reported 24 axons in applicants' aforementioned co-pending applications. Exons 6, 14 and 17, as previously reported, are Bound to be in segments and are now named axons 6a, 6b, axons 14a, 14b and axons 17a, 17b.
The in~tron portions, which have been used in PCR
amplification, are identified in the following Table 5 and underlined in Figure 18. The portions identified by the arrows are selected, but it is understood that other portions of the intron,sequences are also useful in the PCR amplification technique. For example, for axon 10 vy0 91/10734 PCf/CA,91/00009 65 ~~'j~ ~~b~!
a.
the relevant genetic information which is preferd~ed in PCR is noted by reference to the 5° and 3' ends of the sequence. The intron section is identified with an "i".
Hence in Table 5 for axon 2, the preferred portions are identified by 2i-5 and 2i-3 and similarly for axons 3 through 24. For axon 1, the selected portions include the sequence GGA...AAA for 8115-B and ACA...GTG for IOD.
For axon 13, portions are identified by two sets: 13i-5 and C1-lm and %13B-5 and 13i-3A. (This axon (13) is to large and most practical to be completed in two sections). C1-1M and %13B-5 are from axon sequences.
The specific conditions for PCR amplification of indivisual axons are summarized in the following Table 6 and are discussed in more detail hereinafter with respect to the procedure explained in R.K. Saiki et al, Sci nce 23p:1350 (1965).
These oligonucleotides, as derived from the intron sequence, assist in amplifying by PCR the respective axon, thereby providing for analysis for I~NA sequence alterations corresponding to aautations of the CF gene.
The mutations can be revealed by either direct sequence determination of the PCR products or sequencing the products eloped in plasmid vectors. The amplified axon can also be analyzed by use of gel electrophoresis in the manner to be further described. Tt has been found that tlae sections of the intron for each respective axon are of sufficient length to work particularly well with PCR
technique to provide for amplification of the relevant axon.

wc~ 9aiao7~a ~cr,cA~aio~o9 ~,~~~~~~i.'~
~'ASr~ s ss Oligonuchttdes useQ for amplification of CF gene axons by 1?CR
Exon 1'CR primers; 5'-> 3' Amplired produce (bp) 1 , GGAGTTCACTCACCTAAA (Ells-B) 933 ACACGCCCTCCTCTI°TCGTG (lOD) 2 CCAAATCTGTATGGAGACCA (2i-~ 3?8 TATGTTGCCCAGGCTGGTAT (2i~3) 3 CTTGGGTTAATC't'CtrThGGA (3i~5) 3pg ATTCACCAGAT'i'1'CGTAGT!C (3i-3) 4 TCACATATC~G?'ATGA(4I-5) 438 TT'GTACCAGC'I~CACTAG~fA (4i-3) ATTTCTGCCTAGATGCTGGG (Si-,5~ 395 AACTCCGCC'I°T"TCCAGTl'GT (5i-3) ~ TTAGTGTGC1°CAGAACCACG (bAi-~ 385 CTATGaCATAGAGCAGTCC1~ (tSAi-3) 6'b TGGAATGAGTCTGTACAGCG (6Ci-5) 41?
GAGGTGGAAGTCTACCATGA (6Ci-3) ? AGACCATGCTCAGATC'ITCCAT (7i-5) 410 GCAAAGTTCATTAGAACTGATC (7i-3) ' 8 TGAATCCrAGTGCTTGGCAA (8i~~ 3S9 TCGCCATTAGGATGAAATCC (8I-3) 9 TAA'IY~GATCATGGGCCATGT (9i-5) ~p ACA~AATGTGGTGGA (9i-3) GCAGAGTACCTGAAACAGGA (l0i-5) 491 CAT'TCACAGTAGCTTAG'CCA (l0i-3) 11 CAAL'11GTGOTTAAAGCAATAG'lY3T (a li~3) 425 GCACAGATTCTCiAGTAACCATAAT (1li~3) 12 GTGAA'hCpA7T3TGGTCiACCA (12i-5) 426 C~T'ITAGCATOAGGC(3G1° (121.3) I3 (a) TGCTAAAATACGA~ACATATTGCA (13i-5) 52g A'd~.°I'CrGI°AC'I'AAGCiACA(3 (Cl-1M) (b) TCAATCCAATCAAC'PCTATACGAA (X138-5) 49?
TACACCTTATCCTAATCCTATGAT (13i-3A) 14a AAAAGG1'ATi3CCAL'n31'1'AAG (l4Ai-5) 511 GTATACATCOa',AAAC1°ATCT (l4Ai-3) .
14b GAACALXTAGTACAGCT~1' (idii;i~5) 449 AAC'lY~'1~GGGC'rCleAf3TGAT (14BI-3) GTGCATG~CZ°!'C1'AA'lYiCA (15I-5) 485 AAGGCACaITGt~CTC'1°GTtiCA (15i~3) 16 CAGAGAAATTGGT~r °TACT (16i-~ 5?p A'I'~."1'AAAGGA1'~T (16i~3) 1?s ' CAA,'PC3TGCACA'hCiTAt~C'TA (1?AI-5) 5?9 TGTACACCAAC'1°GTGGTAAG (1?Ai-3) 1?b TTCAAAGAATGGCACCAGTGT (17BI-5) 463 ATAACGTATAGAATGCAGCA (1?Bl-3) 18 GTAGATGCTGTC1ATGAAG'TQ (18i~5) 451 AGTraGCTATGTATGAGAAGG (18i~3) 19 GQOCGACAAATAACCAAGTGA (19I-5) ~ 454 GCTAACACATTGCT1'CAGGC'T (19i~3) GG"1T,'AGGATTGAAAGTGTGCA (1~OI-5) 4?3 C'd'ATGAGAAAACTGCAChCiGA (?Gi-3) 21 AATGTTCACAA~iGGACTCCA (21i-~ 4?7 GTACCTGT1'GC'1'CCA (21i-3) ~ AAAACAAGA (22i-5) 562 TGTCAt7CATGAAGCAGGCAT (22i~3) 23 AGC'nGAT'hC,iTGCGTAAG'GC'1' (23i-:~ 4~p TAAAGCT~CiGATATG (23i-3) 24 GGACACAGCAGTTAMTGTO (24i-5) 5~
ACTATTGCCAGGAAGCCATT (2aii-3) -~ ~-~ ~' E .

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fVO 93/10734 F'CT/Cf9,91/00009 Fr Y _.
,~~~~"~~.~ 68 3.~ CF' MZJ~'~1'I°TOIds ~ AZS~6 O'7~3 Z~507 The association of the F508 deletion with 1 common and 1. rare CF haplotype provided further insight into, the number of mutational events that could contribute to the present patient population, Based on the extensive haplotype data, the original chromosome in which the F508 deletion occurred is likely to carry the haplotype -AAAAAAA- (Group Ia), as defined in Table 4. The other Group I CF chromosomes carrying the deletion are probably recombination products derived from the original chromosome. If the CF chromosomes in each haplotype group are considered to be derived Pram the same origin, only 3-4 additional mutational events would be predicted (see Table 4). However, since many of the CF chromosomes Z5 in the same group are markedly different from each other, further subdivision within each group is possible. As a result, a higher number of independent mutational events could be considered and the data suggest that at least 7 additional, putative mutations also contribute to the CF-PI phenotype (see Table 3). The mutations leading to the CF-PS subgroup are probably more heterogeneous.
The 7 additional CF-PI mutations are represented by the haplotypes: -CAAAAAA- (Group Ib), -CABCAAD- (Group I~), ___BBBAC- (Group IIa), -cASBaAB- Group va).
Although the molecular defect in each of these mutations has yet to be defined, it is clear that none of these mutations severely affect the region c~rresponding to the oligonucleotic~e binding sites used in the PCR/hybridization experiment.
One CF chromosome hydridizing to the ~F508-ASO
probe, however, has been found to associate with a different haplotype (group IIIa). It appeared that the ~F508 should have occurred in both haplotypes, but with the discovery of aI507, it is discovered that it is not.
Instead, the 0F508 is in group Ia, whereas the ~I50? is in group IIIa. None of the other CF nor the normal chromosomes of this haplotype group (IIIa)have shown WO 91 / 10734 PCT/Cr191 /00009 n .~ r a Il :f l'~ .~ r'4 ~1 hybridization to the mutant (~F508) ASO [B. Kerem et al, Science 245:1073 (1989)x. In view of the group Ia and IIIa haplotypes being distinctly different from each other, the mutations harbored by these two groups of CF
chromosomes must have originated independently. To investigate the molecular nature of the mutation in this group IIIa CF chromosome, we further characterized the region of interest through amplification of the genomic DNA from an individual carrying the chromosome IIIa by the polymerase chain reaction (PCR).
These polymerase chains reactions (PCR) were performed according to the procedure of R.K. Saiki et al Science 230:1350 (1985). A specific DNA segment of 491 by including axon l0 of the CF gene was amplified with the use of the oligonucleotide primers 10i-5 (5'-GCAGAGTACCTGAAACAGGA-3') and 10i°3 (5'CATTCACAGTAGCTTACCCA-3')located in the 5' and 3' flanking regions, respectively, as shown in Figure 18 and itemzied in Table 5. Eoth oligonucleotides were 2o purchased from the FiBC DNA Biotechnology Service Center (Toronto). Approximately 500 ng of genomic DNA from cultured lymphoblastoid cell lines of the parents and the CF child of Family 5 were used in each reaction. The DNA
samples were denatured at 94°C for 30 sec., primers annealed at 55°C for 30 sec., and extended at 72°C for 50 sea. (with 0.5 unit of Taq polymerise, Perkin-Elmer/Cetus, Norwalk, CT) for 30 cycles and a final extension period of 7 min. in a Perkin-Elmer/Cetus DNA
Thermal Cycler. Reaction conditions for PCR
amplification of other axons are set out in Table 6.
Hydridization analysis of the PCR products from three individuals of Family 5 of group ~IIa was performed. The carrier mother and father are represented by a half-filled circle and square, respectively, and the affected son is a filled square in Figure 19a. The conditions for hybridizaton and washing have been previously described (Kerem et al, su~a). There is a I~VO 91/10734 1'CTJCA91J00009 f '~l~le)~a~~ j 70 relatively weak signal in the father°s PCR product with the mutant (oligo 0F508) probe. In Figure 19b, DNA
sequence analysis of the clone 5-3-15 and the PCR
products from the affected son and the carrier father are shown. The arrow in the center panel indicates the presence of both A and T nucleotide residue in the same position; the arrow in the right panel indicates the points of divergence between the normal and the 0I507 sequence. The sequence ladders shown are derived from the reverse-complements as will be described later.
Figure 19c shows the DNA sequences and their corresponding amino acid sequences of the normal, aI507, and 0F508 alleles spanning the mutation sites are shown.
With reference to Figure 19a, the PCR-amplified DNA from the carrier father, who contributed the group IIIa CF
dhr~mosome to the affected son, hybridized less efficiently with the eF508 ASO than that from the mother who parried the group Ia CF chromosome. The difference became apparent when the hybridization signals were compared to that with the normal As0 probe. This result therefore indicated that the mutation carried by the group IIIa CF chromosome might not be identical to eF508.
To define the nucleotide sequence corresponding to the mutant allele on this chromosome, the PCR-amplified product of the father°s DNA was excised from a polyacrylamide-electrophoretic gel and cloned into a sequencing vector.
The general procedures for DNA isolatian and purification for purposes of cloning into a sequencing vector are described in J. Sambrook, E.F. Fritsch, T.
Maniatis, Molecular Cloni_na~ A I,a a~r~ry gq~~nual, 2nd ed.
(Cold Spring Harbor Press, N.Y. 1989). The two homoduplexes generated by PCR amplification of the paternal DNA were purified from a 5% non-denaturing polyacrylamide gel (30:1 acrylamide:bis-acrylamide). The appropriate bands were visualized by staining with ethidium bromide, excised and elwted in TE (10 mM Tris-4f~ 91110734 PC1'/~A91/00009 71 ~~ ~j~~~~~:d HClp 1mM EDTAa pH 7,5j for 2 to 12 hours at room temperature. The DNA solution was sequentially treated with Tris-equilibrated phenol, phenol/CHC13 and CHClj.
The DNA samples were concentrated by precipitation in ethanol and resuspension in TE, incubated with T4 polynucleotide kinase in the presence of ATP, and ligated into diphosphorylated, blunt-ended Bluescript KS~° vector (Stratagene, San Diego, CA). Clones containing amplified product generated from the normal parental chromosome l0 were identified by hybridization with the aligonucleotide N as described in Kerem et al supra.
Clones cantaining the mutant sequence were identified by their failure to hybridize to the normal ASO (Kerem et al, supra), One clone, 5~3-15 was isolated and its DNA sequence determined. The general protocol for sequencing cloned DNA is essentially as described [3,Rs ~t~,ordan et al, c' a 24511066 (1989)] with the use of an U.S. Biochemicals Sequenase~' kit. To verify the sequence and to exclude any errors introduced by DNA
poly~nerase during PCR, the DNA sequences for the PCR
products from the father and one of the affected children were also determined directly without cloning.
This procedure was accomplished by denaturing 2 pmoles of gel-purified double=stranded PCIt product in 0.2 M NaOH/0.2 mM EDTA (5 min. at room temperature), neutralized by adding 0.1 v~lume of 2 M ammonium acetate (pH 5.4) and precipitated with 2.5 volumes of ethanol at -70°C for 10 yin. After washing with 70~ ethanol, the DNA pellet was dried and redissolved in a sequencing reaction buffer containing 4 pmoles of the oligonucleotide primer 10i-3 of Figure 18, dithiothreitol (8.3 mM) and (a-35S]-dATP (0.8 ACM, 1000 Ci/mmole). The mixture was incubated at 37~C for 20 min., following which 2 ~sl of labelling mix, as included in the Sequenase" Kit and then 2 units of Sequenase enzyme were added. Aliquotes~of the reaction mixture (3.5 ~1) were transferred, without delay, to tubes each containing 2.5 ~1 of ddGTP, ddATP, ddTTP and ddCTP solutions (U.S. Biochemicals SequenaseTM
kit) and the reactions were stopped by addition of the stop solution.
The DNA sequence for this mutant allele is shown in Figure 19b. The data derived from the cloned DNA and direct sequencing of the PCR products of the affected child and the father are all consistent with a 3 by deletion when compared to the normal sequence (Figure 19c). The deletion of this 3 by (ATC) at the 1506 or 1507 position results in the loss of an isoleucine residue from the putative CFTR, within the same ATP-binding domain where OF508 resides, but it is not evident whether this deleted amino acid corresponds to the position 506 or 507. Since the 506 and 507 positions are repeats, it is at present impossible to determine in which position the 3 by deletion occurs. For convenience in later discussions, however, we refer to this deletion as oI507.
The fact that the 0:1507 and OF508 mutations occur in the same region of the presumptive ATP-binding domain of CFTR is surprising. Although the entire sequence of 01507 allele has not been examined, as has been done for OF508, the strategic location of the deletion argues that it is the responsible mutation for this allele. This argument is further supported by the observation that this alteration was not detected in any of the normal chromosomes studied to date (Kerem et al, su ra .
The identification of a second single amino acid deletion in the ATP-binding domain of CFTR also provides information about the structure and function of this protein.
Since deletion of either the phenylalanine residue at position 508 or isoleucine at position DI507 is sufficient to affect the function of CFTR such that it causes CF
disease, it is suggested that these; residues are involved in the folding of the protein but not directly in the binding of ATP. That is, the length of the peptide is probably .~~.,ra..~a aL..~ aL.. ....a....7 .....,......, ....:.-7 'W~ 91/10734 PCT/CA91/00009 ~~.~.)'~ l; .3 ~d Cf ~a res;dues in this region. In support of this hypothesis, it has been found that the phenylalanine residue can be replaced by a serine and that isoleucine at position 506 with valine, without apparent loss of function of CFTR.
When the nucleotide sequence of dT507 is compared to that of aF5O8 at the ASO-hybridizing region, it was noted that the difference between the two alleles was only an A
-~ T change (Figure 19c). This subtle difference thus explained the cross-hybridization of the dF508-ASO to to 0I507. These results therefore exemplified the importance of careful examination of both parental chromosomes in performing ASO-based genetic diagnosis.
it has bean determined that the eF5o8 and nT507 mutations can be distinguished by increasing the stringency of oligonucleotide hybridization condition or by detecting the unique mobility of the heteroduplexes formed between each of these sequences and the normal DNA on a polyacrylaminde gel. The stringency of hybridization can be increased lay using a washing temperature at ~5°C
instead of the prior 39°C in the presence of 2XSSC (1XSSC
- 25,0 mM NaCL and 15 mM Na citrate).
Identification of the ~I507 and eF508 alleles by polyacrylamide gel electrophoresis is shown in Figure 20.
The PCR products were prepared from the three family members and separated on a 5~ polyacrylamide gel as described above. A DNA sample from a known heterozygous nF508 carrier is included for comparison. With reference to Figure 20, the banding pattern of the PCIt-amplified genomic DNA from the father, who is the carrier of nI507, is clearly distinguishable from that of the mother, who is of the type of carriers with the aF508 mutation. In this gel electrophoresis test, there were actually three individuals (the carrier father and the two affected sons in Family 5) who carried the ~iI507 deletion. Since they all belong to the same family, they only represent one single CF chromos~me in our population analysis [Kerem et al, supra] The two patients who also inherited the nF508 WO 9111a73~t &'C'1'/CA91/00009 ~~ ~ '~3~.y y mutation from their mother showed typical symptoms of CF
with pancreatic insufficiency. The father of this family was the only parent who carries this QI507 mutation; no other CF parents showed reduced hybridization intensity signal with the eF508 mutant oligonucleotide probe or a peculiar heteroduplex pattern for the PCR product (as defined above) in the retrospective study. In addition, two representatives of the group IIIb and one of the group IIIc CF chromosomes from our collection [Kerem et l0 a1, sutra] were sequenced, but none were found to contain eI507. Since the electrophoresis technique eliminates the need for probe-labelling and hybridization, it may prove to be the method of choice for detecting carriers in a large population scale [J. M. Rommens et al, Am. J.
Hum. Genet. 46:395-396 (1990)x.
The present data also indicate that there is a strict correlation between DNA marker haplotype and mutation in CF. The ~F508 deletion is the most common CF
mutation that odcurred on a group Ia chromosome background [Kerem et.al, sutara~. The pI507 mutation is, however, rare in the CF population; the one group~IIIa CF
chromosome carrying this deletion is the only example in our studied population (1/219). Since the group III
haplotype is relatively common among the normal chromosomes (17/198), the ~I507 deletion probably occurred recently. Additional studies with larger populations of different geographic and ethnic backgrounds should provide further insight in understanding the origins of these mutations.
3 0 3~8 ~?DITIONAL C~ MtIT~TTCNB
Following the above procedures, other mutations in the CF gene have been identified. The following brief description of each identified mutation is based on the previously described procedures for locating the mutation involving use of PCR procedures. The mutations are given short form names. The numbering used in these abbreviations refers to either the DNA sequence or the.

r3'0 g1!10734 PC1'/CA91/OtD009 75 ~~~u mLi~
amino acid sequence position of the mutation depending on the type of mutation. For example, splice mutations and frameshift mutations are defined using the DNA sequence position. Most otrar mutations derive their nomenclature from the amino acid residue position. The description of each mutation clarifies the nomenclature in any event.
For example, mutations 6542X, Q493X, 3659 dal C, 556 dal A result in shortened polypeptides significantly different from the single amino acid deletions or alteration. 6542X and Q493X involve a pnlypeptide including on the first 541 and 493 amino acid residues, respectively, of the normal 1480 amino acid polypeptide.
3659 dal C and 556 dal A also involve shortened versions and will include additional amino acid residues.
Mutation 711+1G -~ T and 171?-1G ~ A are predicted to lead to polypeptides which cannot b~ as of yet exactly def3.ned. They probably do lead to shortened polypeptides but could contain additional amino acids. DNA sequence encoding these mutant polypeptides will now probely contain intros sec,~aence from the normal gene or possible deleted axons.
3 ~ 8 . 0 ~i,~'ATLQ~T.$ ~H ~~OTJ 1 In the 1296 -~ C mutation, there is a single basepair change of G to C at nucleotide 129 of the cDNA sequence of Figure 1. The PCI2 product for amplifying genomic DNA
containing this mutation is derived from the 8115-B and lOD primers as set out in Table 5. The genomic DNA is amplified as per the conditions of Table 6.
~~z~N~ zr~ ~~oN ~
The G85E mutation in exon 3 involves a G to A
transition at nucleotide position 386. It is detected in family X26, a French Canadian family classified as PI.
This predicted Gly to Glu amino acid change is associated with a group IIb haplotype. The mutation destroys a F3inf1 site. The PCTd product derived from the 3i-5 and 3i-3 primers, as per conditions of Table 6, is cleaved by this enzyme into 3 fragment, 172, 105 and 32 bp, WO 91/1073~t PCT/CA91/00009 resp~ctivelyP for the normal sequence; a fragment of 277 by would be present for the mutant sequence. We analyzed 54 CF chromosomes, 8 from group II, and 50 normal chromosomes, 44 from group II, and did not find another example of G85E.
~.~.a a~~~~xxoN~ xN ~xoN ~
556 dal A is a frameshift mutation in axon 4 in a single CF chromosome (Toronto family X17, GM1076). There is a deletion of A at nucleotide position 556. This mutation is associated with Group IIIb haplotype and is not found in 31 other CF chromosomes (9 from IIIb) and 30 N chromosomes (16 from IIIb). The muation creates a BglI
1 enzyme cleavage site. The PCR primers are 4i-5 and 4i-3 (see Table 5) where the enzyme cuts the mutant PCR
product (437 bp) into 2 fragments of 287 and x,50 by in size.
The I148T mutation in axon 4 involves a T to C
basepair transition at nucleotide position 575. This results in an Ile to Thr change at amino acid position a48 of Figure Z. The PCR product used in amplifying genomic DNA containing this mutation uses primers 4i-5 and 4i-3 as set out in Table 5. The reaction conditions for amplyfing the genomic DNA are set out in Table .6.
3.8.3 M~TATxtaNS ',,~i O~
In mutation G178R the Gly to Arg missense mutation in axon 5 is due to a G t~ A change at nucleotide position 664. The mutation is found on the mother s CF
chromosome in family X50; the other mutation in this family is OF508. primers 5i-5 and 5i-3 were used for amplifying genomic DNA as outlined in Tables 5 and 6.
a. a ~t~r~~xoNS xN ~xo~a~ ~
A mutation in axon 9 is a change of alanine (GCG) to glutamic acid (GAG) at amino acid position 455 (A455 -~ E). Two of the 38 non-eF508 CF chromosomes examined carries this mutation; both of them are from patients of a French-Canadian origin, which we have identified in our work as familie;~ X27 and X53, and they VV~ 91/~~?734 PCT/CA91/OU009 '.
7 7 .x .
belong to haplotype group Ib. The mutation is detectable by allele-specific oligonucleotide (ASO) hybridization with PCR-amplified genomic DNA sequence. The PCR primers are 91-5 (5'-TAATGGATCATGGGC~:ATGT-3') and 9i-3 (5'-ACAGTGTTGAATGTGGTGCA-3') for amplifying genomic DNA under the conditions of Table 6. The ASOs are 5'-GTTGTTGGCGGTTGCT-3~ for the normal allele and 5'-GTTGTTGGAGGTTGCT-3' for the mutant. The oliganucleotide hybridization is as described in Kerem et al (1989) su ra at 37°C and the washings are done twice with SXSSC for 10 min each at room temperature followed by twice with 2 X
SSC for 30 min each at 52°C. Although the alanine at position 455 (A1a455) is not present in all ATP-binding folds across species, it is present in all known members of the P-glycoprotein family, the protein most similar to CFTR. Further, A455 -~ E is believed to be a mutation xat3ier than a sequence polymorphism because the change is not found in 16 non-oF5o8 CF chromosomes and three normal chromosomes carrying the same group I haplotype.
2 0 3 . $ . 5 MDT~S'C'I~1~T8 IId EXON ~( O
In the Q493X mutation G1n493 (CAG) is changed into a stop codon (TAG) in Toronto family ~'9 (nucleotide position 1609 C -~ T). The muation occurs on a CF
chromasome with haplotype IIIbe it is not found in 28 normal chromosomes (15 of which belong to 11b) nor in 33 other CF chromosomes (5 of which IIIb). The mutation can be detected by allele-specific PCR, with 10i-5 as the common PCR primer, 5'-GGCATAATCCAGGAAAACTG-3' for the normal sequence and 5'-GGCATAATCCAGGAAAACTA-3' for the mutant allele. The PCR condition is 6 min at 94°
followed by cycles of 30 sec at 94°, 30 sec at 57° and 90 sec at 72°, with 100 ng of each primer and -40o ng genomic DNA. The primers 9i-3 and 9i-5 may be used for internal PCR control as they share the same reaction condition.
3.8.6 MUTATIONS IN E9COIV 11 WO 9,1/10734 P~1'1CA91100009 In mu~catian G542X the glycine radon (GGA) at amino acid position 542 is changed to a stop colon (TGA) (G542 Stop). The single chromosome carrying this mutation is of Ashkenazic Jewish origin (family A) and has the B
haplotype (XV2C allele 1; KM.19 allele 2). The mutant sequence can be detected by hybridization analysis with allele-speoi~ic oligonucleotides (ASOs) on genomic DNA
amplified under conditions of Table 6 by PCR with the 11i-5 and 11i-3 oligonucleotide primers. The normal ASO
is 5'-ACCTTCTCCAAGAACT-3° and the mutant ASO, 5°-ACCTTCTCAAAGAACT-3°. The oligonucleotide hybridization condition is as described in Kerem et al (1989) su ra and the washing conditions are twice in 5 x SSC for 10 min.
each at room temperature followed by twice in 2 X SSC for 30 min. each at 45°C. The mutation is not detected in 52 other non-ef508 CF chromosomes, 11 of which are of Jewish vr~,c~a.n (three hays a B haplotype), nor in 13 normal chrom~somes.
In mutation S549R, the highly conserved serine residue of the nucleotide binding domain at position 549 is changed to arginine (5549 -~ R);,the colon change is AGT -~ AGG. The CP.chromosome with this mutation is carried by a non-Ashkenazic Jewish pateitn from Morocco (family B). The chromosome also has the B haplotype.
Detection of this mutation may be achieved by ASO
hybridization or allele-specific PCR. In the ASO
hybridization procedure, the genomic DNA sequence is first amplified under conditions of Table 6 by PCR with the 11i-5 and 11i-3 oligonucleotides; the ASO for the normal sequence is 5°-ACACTGAGTGGAGGTC-3' and that for the mutant is 5°-ACACTGAGGGGAGGTC. The oligonucleotide hybridization condition is as described by Kerem et al (1989) supra and the washings are done twice in 5 x SSC
for 10 min. each at room temperature followed by twice in 2 x SSC for 30 min. eachat 56°C. In th.e allele-specific PCR amplification, the,oligonucleotide primer for the normal sequence is 5'TGCTCGTTGACCTCCA-3', that for the WO 91/10731! ~ n ,7 ~ ;Q .~ :; PCTlCA91/00009 7~
mutant is 5°TGCTCGTTGACCTCCC-3° and that fir the common, outside sequence is 11i-5. The reaction is performed With 500 ng of genomie DNA, 100 ng of each of the oligonucleotides and 0.5 unit of Taq polymerase. The DNA
template is first denatured by heating at 94°C for 6 min., followed by 30 cycles of 94° for 30 sec, 55° for 30 sec and 72° for 60 sec. The reaction is completed by a 6 min heating at ?2° for 7 min. This 5549 -» R mutation is not present in 52 other non-oF508 CF chromosomes, 11 of which are of Jewish origin (three have a B haplotype), nor in 13 normal chromosomes.
In the S549I mutation there is an AGT~ATT change (nucleotide position 1778 G-~T) which represent the third mutation involving this amino acid colon resulting in a loss of the Ddel s3.te. We have only one example who is of Arabic origin and is sequenced; no other Ddel-resistant chromosome is found in 5 other Arabic CF, 21 Jewish CF, 41 Canadian CF, and 13 Canadian normal chromosomes.
In mutation R560T the arginine (AAG) at amino acid position 560 is changed to threonine (AAC). The individual carrying this mutation (R560 -~ T) is from a family we have identified in our work as family X32 and the chromosome is marked by haplotype IIIb. The mutation creates a MaeII site which cleaves the PCR product of axon 11 (generated with primers !1i-5 and !1i-3 under oonditions of Table 6) into two fragments of 214 and 204 by in size. None of the 36 non-~F508 CF chromosomes (seven of which have haplotype IIIb) or 23 normal chromosomes (16 have haplotype IIIb) carried this sequence alteration. The 8560 w T mutation is also not present on eight CF chromosomes with the ~F508 mutation.
In mutation G551,D glycine (G) at amino acid position 551 is changed to asgartic acid (D). 6551 is a highly conserved residue within the ATP-binding fold. The corresponding colon change is from GGT to GAT. The 6551-~D change is found in 2 of our families (~'1, X38) '1'~~ 91110734 PC1'/CA91/00009 ~3~~~~~'-,~~~~ ' ~0 with pancreatic .insufficient (PI) CF patients and 1 family (#54) with a pancreatic sufficient (PS) patient.
The other CF chromosomes in family #i and X38 carry the AF508 mutation and that in family #54 is unknown. Based on our °°severe and mild mutation°' hypothesis (Iterem et al. 1989), this mutation is expected to be a "severe"
one. All 3 chromosomes carrying this mutation belong to Group IIIb. This 6551-~D substitution does not represent a sequence polymorphism because the change is not detected in 35 other CF chromosomes without the eF508 deletion (5 of them from group IIIb) and 19 normal chromosomes (including 5 from.group IIIb). To detect this mutation, the genomic DNA region may be amplified under conditions of Table 6 by PCR with primers 11i-5 (5'-CAACTGTGGTTAAAGCAATAGTGT-3') and 11i--3 (5~-GCACAGATTCTGAGTAACCATAAT-3') and examined for the presence of a Mbol (Sau3A) site created by nucleotide change; the uncut (normal) form is 419 by in length and the digestion products (from the mutant form) are 241 and 178 bp.
.3 ~ ~ . 7 ~f»T~ I~1 EI~Id 3.2 ' Zn the Y563N mutation a T to A change is detected at nucleotide position 1820 in exon 12. This switch would result in a change from Tyr to Asn at amino acid position 563. It is found in a single family with 2 PS patients but the mutation in the other chromosome is unknown. We think Y563N is probably a missense mutation because (1) the T to A change is not found in 59 other CF
chromosomes, with 8 having the same haplotype (IIa) and 30 having vF508; and (2) this alteration is not found in 54 normal chromosomes, with 39 having the 11a haploytype.
Unfortunately, the amino acid bhange is not drastic.
enough to permit a strong argument. This putative mutation can be detected by ASO hybridization with a normal (5°-AGCAGTATACAAAGATGC-3') and a mutant (5~-AGCAGTAAACAAAGATGC-3~) oligonucleotide probe. The washing condition is 54°C with 2xSSC.

WO 91/10734 PCT/CA91/~D0009 cy~ w1 :a .S .;
~a ~ d c7 ' ~' ~:.:

In the P574H mutation the C at nucleotide pasi,~ion 1853 is changed to A. Although the amino acid Pro at this position is not highly conserved across different ATP-binding folds, c change to His could be a drastic substitution. This change is not detected in 52 ether CF
chromosomes nor 15 normal chromosomes, 4 of which have the same group IV haplotype. Based on these arguments, we believe P574H is a mutatian. To detect this putative mutation, one may use the following ASOs: 5'-l0 GACTCTCCTTTTGGA-3' far the normal and 5°-GACTCTCATTTTGGA-3° for the mutant. Washing should be dune at 47~ in 2xSSC.
In the L1077P mutation, the T at nucleotide position 3362 is changed to C. This results in a change of the amino acid Leu to Faro at amino position 1.077 in Figure 1.
As with the other mutations in this axon, the genomic DNA
is amplified by use of the primers of Table 5; namely l7bi-5 and 1?bi-3. The reaction conditions in amplifying the genamic DNA are set out in Table 6.
The Y1092x mutation involves a change of C at nucleatide p~sition 3408 to A. This would result in protein synthesis termination at amino position 1092.
Hence the amino acid Tyr is not present in the truncated polypeptide. As with the above procedures, the primers used in amplifying this mutation are i7bi-3 and l7bi-3.
3 s ~ W ~~a~N~ Z~ ~r~~~ Z
3659 dal C is a frameshift mutation in axon 19 in a single CF chramasome (Taronta family ~2); deletian of C
at nucleatide position 3659 or 3960; haplotype IIa; not present in 5? non-aF508 CF chromosomes (7 from IIa) and 50 N chromosomes (43 from IIa); the deletion may be detected by PCR with a common oligonucleatide primer 191-5 (see Table 5) and 2 ASO primers, HSCB (5°-GTATGGTTTGGTTGACTT GG-3°) far the normal and HSC9 (5°-GTATGGTTTGGTTGACTTGT-3°) for the mutant allele; the PCR
condition is as usual except the annealing temperature is at 60~C to improve specificity.

W~ 91/10734 PCI'/C/e91/000~9 ~flr~~~.~~~.~ 82 3.8.9 ,'MtJ°x°A!~°xCNB IN Il~fiTRON 4 In the 621 + 1G -~ T mutation there is a single by change affecting the splice site (GT -~ TT) at the 3' end of axon 4; this mutation is detected in 5 French-Canadian CF chromosomes (one each in Toronto families X22, 23, 26, 36 and 53) but not in 33 other CF chromosomes (18 from the same group, group I) and 29 N chromosomes (13 from group I); the mutation creates a Msel site; genomic DNA
may be amplified by the 2 intron primers, 4i-5 adn 4i-3, 20 and cut with Msel to distinguish the normal and mutant a11e1es; the normal would give 4 fragments of 33, 35, 7Z
and 298 by in size; the 298 by fragment in the mutant is cleaved by the enzyme to give a 54 and 244 by fragments.
3.8. AO ~QTATIO~TB IN INTRON ;Z
In the 711 + 1.G ~ T mutation this G to T switch occurs at the splice junction after axon 5. The mutation is found ~n the mother's CF chromosome in family ,~22, a French Canadian family from Chicoutimi. The other mutation in this family is 621+iG -~ T.
2 0 ~QTA~'gONB IN INI,~_ON 10 In the 9.73.7-1G -~ A mutation a putative splice mutation is found in front of axon 7.1. This mutation is located at the last nucleotide of the intron before axon 11. The mutation may be detected with the following ASO's> normal ~~ 5'-TTTGGTAATAGGACATCTCC-3'; mutant ASO =
5'-TTTGGTAATAAGACATGTCC-3'. The washing conditions afar hybridization are 5xSSC twice for 10 min at room temp, 2xSSC twice for 30 min at 47° for the mutant and 2xSSC
twice to 30 min at 48° for the normal ASO. We have only 1 single example from an Arabic patient and there is no haploytpe data. The mutation is not found in 5 other Arabic, 21 Jewish, and 41 Canadian CF chromosomes, nor in 13 normal chromosomes.
3.9 DNA SRQtTLNCR 1~OL5tMORP~fIBM~
Nucleotide position Amino acid change 1540 (A or G) Met or Val 1716 (G or A) no change (Glu) 2694 (T or G) no change (Thr) W(~ 91J10734 PCTJCA91l00009 8 3 r.. ~S 9 _s .,..
356 (G or A) Arg or Gln A polymorphism is detected at nucleotide position 1540-the ~. residue can be substituted by G, changing the corresponding amino acid from Met to ~7a1. At postion 2694- the T residue can be a G; although it does not change the encoded amino acid. The polymorphism may be detected by restriction enzymes AvaII or Sau9GI. These changes are present in the normal population and show good correlation with haploytpes but not in GF disease.
There can be a G to A change for the last nucleotide of exan 10 (nucleotide position 1716). We think that this nucleotide substitution is a sequence polymorphism because (a) it does not alter the amino acid, (b) it is unlikely to cause a splicing defect and (c) it occurs on some normal chromosomes. In two Canadian families, this rare allele is found associated with haplotype IIIb.
fihe more common mucleotide at 356 (G) is found to be changed to A in the father's normal chromosome in family ~'~4. The amino acid changes from Arg to Gln.
2 0 ~ C,E~ ~~tMP~I~J
As discussed~~aith respect to the DNA sequence of Figure 1, analysis of the sequence of the overlapping cDNA clones predicted an unprocessed polypeptide of 1480 amino acids with a molecular mass of 166,138 daltons. As later described, due to polymorphisms in the protein, the molecular weight of the protein can vary due to possible substitutions or deletion of certain amino acids. The molecular weight will also change due to the addition of carbohydrate units to form a glycoprotein. It is also 3d understood that the functional protein in the cell will Wa 9i/IU7~t i'C'T/CA91/00409 ....' be aimiiar to the unprocessed polypeptide, but may be modified due to cell metabolism.
Accordingly, purified normal CFTR polypeptide is characterized by a molecular weight of about 170,000 daltons and having epithelial cell transmembrane ion conductance activity. The normal CFTR polypeptide, which is substantially free of other human proteins, is encoded by the aforementioned DNA sequences and according to one embodiment, that of Figure 1. Such polypeptide displays the immunological or biological activity of normal CFTR
polypeptide. As will be later discussed, the CFTR
polypeptide and fragments thereof may be made by chemical or enzymatic peptide synthesis or expressed in an appropriate cultured cell system. The invention provides purified 507 mutant CFTR polypeptide which is characterized by cystic fibrosis-associated activity in human epithelial cells. Such 507 mutant cFTR
polypeptide, as substantially free. of other human proteins, can be encoded by the 50T mutant DNA sequence.
2 0 ~ ST130~°iJRB O~ t:F'fR
The most characteristic feature of the predicted protein is the presence of two repeated motifs, each of which consists of a set of amino acid residues capable of spanning the membrane several times followed by sequence resembling consensus nucleotide (ATP)-binding folds (NBFs) (Figures 11, 12 and 15). These characteristics are remarkably similar tc those of the mammalian multidrug resistant P-glycoprotein and a number of other membrane-associated proteins, thus implying that the predicted CF gene product is likely to be involved in the transport of substances (:tons) across the membrane and is probably a member of a membrane protein super family.
Figure 13 is a schematic modal of the predicted CFTR
protein. xn Figure 13, cylinders indicate membrane spanning helices, hatched spheres indicate NBFs. The stippled sphere is~the polar R-domain. The 6 membrane spanning helices in each half of the molecule are w~ ~~rio7~a Pcrrc~.~nroooo9 ;~ ~ ~ ~~ S : j :, depicted as cylinders. The inner cytoplasmically oriented FdBFs are shown as hatched spheres with slots to indicate the means of entry by the nucleotide. The large polar R-domain which links the two halves is represented by an stippled sphere. Charged individual amino acids within the transmembrane segments and on the R-domain surface are depicted as small circles containing the charge sign. Net charges on the internal and external loops joining the membrane cylinders and on regions of the NBFs are contained in open squares, Bites for phosphorylation by protein kinases A or C are shown by closed and open triangles respectively. K,R,H,D, and E
are standard nomenclature fox the amino acids, lysine, arginine, histidine, aspartic acid and glutamic acid respectively.
Each of the predicted membrane-associated regions of the CFTR protein consists of 6 highly hydrophobic serpents capable of spanning a lipid bilayer according to the algorithms of Kyte and Doolittle and of Garnier et al 2g (J. Mal. Bio' 1Z0, 97 (1978) (Figure 13). The membrane-associated regions are each followed by a large hydrophilic region containing the NBFs. Based on sequence alignment with other known nucleotide binding proteins, each of the putative NBFs in CFTR comprises at least 15o residues (Figure 13). The 3 by d~letion at position 507 as detected in CF patients is located between the 2 most highly conserved segments of the first NBF in CFTR. The amino acid sequence identity between the region surrounding the isoleucine deletion and the 30. corresponding regions of a number of other proteins suggests that this region is of functional importance (Figure 15), A hydrophobic amino acid, usually one with an aromatic aide chain, is present in most of these proteins at the position corresponding to I507 of the CFTR protein. It is understood that amino acid polymorphi~ms may exist as a result of DNA polymorphisms.
Similarly, mutations at the other positions in the ~;

protein suggested that corresponding regions of the protein'are also of functional importance. such additional mutations include substitutions of:
i) Glu for Gly at amino acid position 85;
ii) Thr for Ile at amino acid position 148;
iii) Arg for Gly at amino acid position l?8;
iv) Glu for ALA at amino position 455;
v) stop colon for Gln at amino acid portion 493;
vi) stop colon for Gly at amino acid position 542;
vii) Arg for Ser or Ile for Ser at amino acid position 549;
viii) Asp for Gly at amino acid position 551;
ix) Thr for Arg at amino acid position 560;
x) Asn for Tyr at amino acid position 563;
xi) His for Pro at amino acid position 574;
xii) Pro for Leu at amino acid position 1077;
xiii) Stop colon for Tyr at amino acid position 1092.
Figure 15 shows alignment of the 3 most conserved segments of the extended NBF's of CFTR with comparable regions of other proteins. These 3 segments consist of residues 433-473, 488-513, and 542-584 of the N-terminal half and 1219-1259, 1277-1302, and 1340-1382~of the C-terminal half of CFTR. The heavy overlining points out the regions of greatest similarity. Additional general homology can be seen even without the introduction of gaps.
Despite the overall symmetry in the structure of the protein and the sequence conservation of the NBFs, sequence homology between the two halves of the predicted CFTR protein is modest. This is demonstrated in Figure 12, where amino acids 1-1480 are represented on each axis. Lines on either side of the identity diagonal indicate the positions of internal similarities.
Therefore, while four sets of internal sequence identity can be detected as shown in Figure 12, using the Dayhoff scoring matrix as applied by Lawrence et aZ. [C. B.
°~~°f IJTT.

WO 91/10734 PC'd'/CA97/00009 ~~i~ a a Lawrence, D. ~. Goldman, and R. T.. Hood, Bull Math liol 48, 569 (1986)], three of these are only apparent at low threshold settings for standard deviation. The strongest identity is between sequences at the carboxyl ends of the NBFs. Of the 66 residues aligned 27% are identical and another 11% are functionally similar. The overall weak internal homology is in contrast to the much higher degree (>70%) in P~glycoprotein for which a gene duplication hypothesis has been proposed (taros et al, Ce 47, 371, 1986, C. Chen et al, C~11 47, 381, 1986, Gerlach et al, Nature, 324, 485, 1986, taros et al, Mol.
Celt. Biol. 8, 2770, 1988). The lack of conservation in the relative positions of the axon-intron boundaries may argue against such a model far CFTR (Figure 2).
15. Since there is apparently no signal-peptide sequence at the amino-terminus of CFTR, 'the highly charged hydrophilic segment preceding the first transmembrane sequence is probably oriented in the cytoplasm. Each of the 2 sets of hydrophobic helices are expected to form 3 transversing loops across the membrane and little sequence of the entire protein is expected to be exposed to the exterior aurtace, except the region between transmembrane segment 7 and 8. It is of interest to note that the latter region contains two potential sites for N-linked glycosylation.
Each of the membrane-associated regions is followed by a NBF as indicated above. xn addition, a highly charged cytoplasmic domain can be identified in the middle of the predicted CFTR polypeptide, linking the 2 halves of the protein. This domain, named the R-domain, is operationally defined by a single large axon in which 69 of the Z41 amino acids are polar residues arranged in alternating clusters of positive and negative charges.
Moreover, 9 of the 10 consensus sequences required for phosphosphorylation by protein kinase A (PxA), and, 7 of the potential substrate sites for protein kinase C (PRC) found in CFTR are located in this axon.

bV~ 91/10734 P~'/CA91/00009 'J s.~ s . n .
~~ ~ ~l~~L a . s8 FuzaCTlC~la of CFfiR
Properties of CFTR can be derived Pram comparison to other membrane-associated proteins Figure 15). In addition to the overall structural similarity with the mammalian P-glycoprotein, each of the two predicted domains in CFTR also shows remarkable resemblance to the single domain structure of hemolysin B of ,~ Coli and the product of the White gene of Drosophila. These fatter proteins are involved in the transport of the lytic 2o peptide of the hemolysin system and of eye pigment molecules, respectively. The vitamin B12 transport system of ,~,,. cc~li, BtuD and MbpX which is a liverwort chloroplast gene whose function is unknown also have a similar structural motif. Furthermore, the CFTR protein Z5 shares structural similarity witty several of the periplasmic solute transport systems of gram negative bacteria where the transmembrane region and the ATP-binding folds are contained in separate proteins which function in concert with a third substrate-binding 20 polypeptide.
The overall structural arrangement of the transmembrane domains in CFTR is similar to sesreral cation channel proteins and some canon-translocating ATPases as well.as the recently described adenylate 25 cyclase of bovine brain. The functional significance of this topological classification, consisting of C
transmembrane damains, remains speculative.
Short regions of sequence identity have also been detected between the putative transmembrane regions of 30 CFTR and other membrane-spanning proteins.
Interestingly, there are also sequences, 18 amino acids in length situated approximately 50 residues from the carboxyl terminus of CFTR and the ref serine/threonine kinase protooncogene of Xeng~ous ~a~yis which are 35 identical at 12 of these positions.
Finally, an amino. acid sequence identity (10/13 conserved residues) has been noted between a hydrophilic ~'O 91/10734 ~ ~;;'y ~ ~ _~ ~ PCT/CA91/U0009 segment (position 701-713) within the highly charged R-domain of CFTR and a region immediately preceding the first transmembrane loop of the sodium channels in both rat brain and eel. The charged R-domain of CFTR is not shared with the topologically closely related R-glycoprotein; the 241 amino acid linking-peptide is apparently the major difference between the two proteins.
In summary, features of the primary structure of the CFTR protein indicate its possession of properties to suitable to participation in the regulation and control of ion. transport in the epithelial cells of tissues affected in.CF. Secure attachment to the membrane in two regions serve to position its three major intracellular domains (nucleotide-binding folds 1 and 2 and the R-domain) near the cytoplasmic surface of the cell membrane where they can modulate ion movement through channels formed either by CFTR transmembrane segments themselves or by other me~abrane proteins.
In view of the genetic data, the tissue-specificity, and the predicted properties of the CFTR protein, it is reasonable to conclude that CFTR is directly responsible for CF. It, however, remains unclear how CFTR is involved in the regulation of ion conductance across the apical membrane of epithelial cells.
It is possible that CFTR serves as an ion channel itself. As depicted in Figure 13, 10 of the 12 transmembrane regions contain one or more amino acids with charged'side chains, a property similar to the brain sodium channel and the GABA receptor chloride channel subunits, where charged residues.are present in 4 of the 6, and 3 of the 4, respective membrane-associated domains per subunit or repeat unit. The amphipathic nature of these transmembrane segments is believed to contribute to the channel-forming capacity of these molecules.
Alternatively, CFTR may not be an ion channel but instead serve to regulate ion channel activities. In support of the latter assumption, none of the purified polypeptides IJS~'~T1'HET

WO 91J1073a fC'f/CA91/00009 'J ~1 9 ~ t~ (. ~3 90 h:
from trachea and kidney that are capable of reconstituting chloride channels in lipid membranes [Landry et al, Science 224:1469 (3.989)) appear to be CFTR
if judged on the basis of the molecular mass.
In either case, the presence of ATP-binding domains in CFTR suggests that ATP hydrolysis is directly involved and required for the transport function. The high density of phosphorylation sites for PKA and PKG and the clusters of charged residues in the R-domain may both l0 serve to regulate this activity. The deletion of a phenylalanine residue in the N5F may prevent proper binding of ATP or the conformational change which this normally elicits and consequently result in the observed insensitivity to activation by PKA- or PKC-mediated phosphorylation of the CF apical chloride canductance pathway. Since the predicted protein contains several doma~.n,s and belongs to a family of proteins which frequently function as parts of mufti-component molecular systems, CFTR may also participate in epithelial tissue functions of activity or regulation not related to ion transport.
With the isolated CF gene (cDNA) now in hand it is possible to define the basic biochemical defect in CF and to further elucidate the control of ion transport pathways in epithelial cells in general. Piost important, knowledge gained thus far from the predicted structure of CFTR.together with the additional information from studies of the protein itself provide a basis far the development of improved means of treatment of the disease, In such studies, antibodies have been raised to the CFTR protein as later described.
S . 0 CF' SCRE~INIIdG
5~1 DNA HABED D%AGNOSIS
Given the knowledge of the 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092 amino acid position mutations and the nucleotide sequence varients at DNA sequence positions 129, 556, 621+1, SI~TI'~!J°~h~ET.

iV0 91/10734 PC'T/CA9y/00009 r~ :~
~'~ :1 ~3 91 ø' ,. c~ 's '., ?
711-1, 1717°1 and 3659 as disclosed herein, carrier screening and prenatal diagnosis can be carried out as follows.
The high risk population for cystic fibrosis is Caucasians. For example, each Caucasian woman andjar man of child°bearing age would be screened to determine if she or he was a carrier (approximately a 5~ probability for each individual). If both are carriers, they are a couple at risk for a cystic fibrosis child. ~aoh child l0 of the at risk couple has a 25% chance of being affected with cystic fibrosis. The procedure for determining carrier status using the probes disclosed herein is as follows.
For purposes of brevity, the discussion on screening by use of one of the selected mutations is directed to the I507 mutation. It is understood that screening can also be accomplished using one of the other mutations or using several of the mutations in ~ screening pr~cess or mutation detection process of this section on CF
2o screening involving DNA diagnosis and mutation detection.
one major application of the DNA sequence information of the normal and 507 mutant CF gene is in the area of genetic testing, carrier detection and prenatal diagnosis. Individuals carrying mutatians in the CF gene (disease carrier or patients) may be detected at the DNA level with the use of a variety of techniques.
The genomic DNA used for the diagnosis may be obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen 34 and autopsy material. The DNA may be used directly for detection of specific sequence or may be amplified enzymatically ,~,n vitro by using FCR [Saiki et al. Science 230: 1350-1353, (1985), Saiki et al. to a 324: 163-3.66 (1986) prior to analysis. RNA or its cDNA form may also be used for the same purpose. Recent reviews of this subject have been presented by Casket', [science 236:
ST~'~ TE SHEEN' WO 91/1073x1 PC.T/CA91/00009 J

1223-8 (1989) and by Landegren et al (Science 242: 229-237 (7.989) ].
The detection of specific DNA sequence may be achieved by methods such as hybridization using specific oligonucleotides [Wallace et al. Cold Spring Harbour Symp. Ouant. Riol. 51: 257-261 (1986)], direct DNA
sequencing [Church and Gilbert, Proc. Nat. Aced. Sci. U.
S. A. 81: 1991-1995 (1988)], the use of restriction enzymes (Flavell et al. Ce 15: 25 (1978), Geever et al Frdc. Nato Acad. Scl,. 'U. S. A. 78: 5081 (1981)], discrimination on the basis of electrophoretic mobility in gels with denaturing reagent (Myers and Maniatis, Cold Spring Harbour Sym. guant. Biol. 51: 275-284 (1986)), RNase protection (Myers, R. M., Larin, J., and T.
Maniatis Science 230: 1242 (1985)), chemical cleavage (Cotton et al Proc. Nat. Aced. Sci. U. S. A. 85: 4397-4401, (2985)) and the ligase-mediated detection procedure [Landegren et al sc'ence 241:1077 (1988)].
Oligonucleotides specific to normal or mutant sequences are chemically synthesized using commercially available machines, labelled radioactively with isotopes (such as 3zP) ar non-radioactively (with tags such as biotin (Ward and Langer et al. Proc. Nat. Ac~,d. Sci. U.
S. A. 78: 6633-6657 (1981)), and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis. The presence or absence of these specific sequences are visualized by methods such as autoradiography or fluorometric (Landegren et al, 1989, supra) or colorimetric reactions (Gebeyehu et a. Nucleic Acids Research 1.5: 4513-4534 (198?)): An embodiment of this oligonucleotide screening method has been applied in the detection of the I507 deletion as described herein.
Sequence differences between normal and mutants may be revealed by the direct DNA sequencing method of Church and Gilbert (su~r~,). Cloned DNA segments may be used~as probes to detect specific DNA segments. The sensitivity SSTi~'T'E ET

W() 91/10734 1"CTlCA91/00009 ~d V ~~ C.) '~ ' t of this method is greatly enhanced when combined with PCR
[Wrichnik et al, Nucleic Ac?d~ ~~e 15:529-542 (1987);
Worig et al, I,~'~~g 330:384-386 (1987)i StOflet et al, 'e c 239:491-494 (1988)x. In the latter procedure, a sequencing primer which lies within the amplified sequence is used with double-stranded PCR product or single-stranded template generated by a modified PCR.
The sequence determination is performed by conventional procedures with radiolabeled nucleotides or b~ automatic sequencing procedures with fluorescent-tags.
sequence alterations may occasionally generate fortuitous restriction enzyme recognition sites which are revealed by the use of appropriate enzyme digestion followed by Eonventional gel-blot hybridization (Southern, J. M01. 8in1 98: 503 (1975)). DNA fragments carrying the site (either normal or mutant) are detected bf their reduction in size or increase of corresponding restriction fragment numbers. Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme; fragments of different sizes are then visualized under W light in the presence of ethidium bromide after gel electrophoresis.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing reagent. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. For example, the PCR product with the 3 by deletion is clearly distinguishable from the normal sequence on an 8% non-denaturing polyacrylamide gel. DNA
fragments of different sequence compositions may be distinguished on denaturing formamidQ gradient gel in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific l~partial-melting~o temperatures (Myers, su~ra)~ In addition, sequence alterations, in particular small deletions, may be~detected as changes in the migration pattern of DNA heteroduplexea in non-denaturing gel electrophoresis, as haws been detected for the 3 by (T507) mutation and in other experimental systems N [Nagamine et al, Am. ~. Hum. Genet, 45:337-339 (1989)).
Alternatively, a method of detecting a mutation comprising a single base substitution or other small change could be based on differential primer length in a PCR. For example, one invariant primer could be used in addition to a primer specific for a mutation. Ths PCR
products of the normal and mutant genes can then be differentially detected in acrylamide gels.
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase (Myers, supra) and S1 protection (Berk, A. J., and P. A.
Sharps Proc. Nat: Aced. Sci. t1: S. A 75: 1274 (1978)), the chemical cleavage method (Cotton, ) or the ligase-mediated detection procedure (Landegren ).
Tn addition to conventional gel-electrophoresis and blot-hybridization methods, DNA fragments may also be visualized by methods where the individual DNA samples are not immobilized on membranes. the probe and target sequences may be both in solution or the probe sequence may be immobilized [Saiki et al, Proc. Natl. Act Wig, 86:6230-6234 (1989)). A variety of detection ~25 methods, such as autoradiography involving radioisotopes, direct detection of radioactive,decay (in the presence or absence ~f scintillant), spectrophotometry involving colorigenic reactions and f.luorometry involving fluorogenic reactions, may be used to identify specific individual genotypes.
Sinee more than one mutation is anticipated in the CF gene such as I507 and F508, a multiples system is an ideal protocol for screening CF carriers and detection of specific mutations. For example, a FCR with multiple, specific oligonucleotide primers and hybridization probes, may be used to, identify all possible mutations at the same time (Chamberlain et al. Nua~eic Acids Research wo ~ar~o73a pcz~rcA9~roooo~
9 5 ~e 16: 1141-1155 (1988)). 'The procedure may invoave immobilized sequence-specific oligonucleotides probes (Saiki et al, supra).
~'2 DFTE(:'fIN(3 TNN Cx' 507 MUTl~'J~"Ic72d These detection methods may be applied to prenatal diagnosis using amniotic fluid cells, chorionic villi biopsy or sorting fetal cells from maternal circulation.
The test for CF carriers in the population may be incorporated as an essential component in a broad-scale genetic testing program for common diseases.
According to an embodiment of the invention, the portion of the DNA segment that is informative for a mutation, such as the mutation according to this embodiment, that is, the portion that immediately surrounds the 2507 deletion, can then be amplified by u~,ing standard PCR techniques [as reviewed in Landegren, Ulf, Robert Kaiser, C. Thomas Casket', and Leroy Hood, DNA
Diagnostics - Molecular Techniques and Automation, in Science 242: 229-237 (1988)x. It is contemplated that the portion of the DNA segment which is used may be a single DNA segment or a mixture of different DNA
segments. A detailed description of this technique now follows.
A specific region of genomic DNA from the person or fetus is to be screened. Such specific region is defined by the oligonucleotide primers C16B
(5'GTTTTCCTGGATTATGCCTGGCAC3') and C16D
(5'GTTGGCATGCTTTGATGACGCTTC3') or as shown in Figure Z8 by primers 101-5 and 10i-3. The specific regions using 10i-5 and 10i-3 were amplified by the polymerase chain reaction (PCR). 200-400 ng of genomic DNA, from either cultured lymphoblasts or peripheral blood samples of CF
individuals and their parents, were used in each PCR with the oligonucleotides primers indicated above. The ,oliganucleotides were purified with Oligonucleotide Purification Cartridges'" (Applied Biosy~tems) or NENSORB'~
PRBP columns (Dupont) with procedures recommended by the WiD 91/10734 PCTlCA91J00009 suppliers. The primers were annealed at 55°C for 30 sec, extended at 72'C for 60 sec (with 2 units of Taq DNA
polymerase) and denatured at 94'C for 60 sec, for 30 cycles with a final cycle of 7 min for extension in a Perkin-Elmer/Cetus automatic thermocycler with a Step-Cycle program (transition setting at 1.5 min). Portions of the PCR products were separated by electrophoresis on 1.4~ agarose gels, transferred to Zetabind"'; (Biorad) membrane according to standard procedures.
The normal and 0I507 oligonucleotid~ probes of Figure 19 (l0 ng each) are labeled separately with 10 units of T4 polynucleotide kinase (Pharmacia) in a 10 ~1 reaction containing 5o mM Tris-HC1 (pH7.6}, 10 mM MgCl2, 0.5 mM dithiothreitol, l0 mrii spermidine, 1 mM EDTA and 30-40 ~eCi of ~r[3ap~ - ATp for 20-30 min at 37°C, The unincorporated radionucleotides were removed with a Sephadex G-25 column before use. The hybridization conditions were as described previously (J.M. Rommens et al Am. J. Hug- Genet. 43,645 (1988}) except that the temperature can be 37'C. The membranes are washed twice at room temperature with 5xSSC and twice at 39°C with 2 x SSC (1 x SSC = 150 mM NaCl and 15 mM Na citrate}.
Autoradiography is performed at room temperature overnight. Autoradiographs are developed to show the hybridization results of genomic DNA with the 2 specific oligonucleotide probes, Probe C normal detects the normal DNA sequence and probe C pI507 detects the mutant sequence.
Genomic DNA sample from each family member can, as explained, be amplified by the .polymerase chain reaction using the intron sequences of Figure 18 and the products separated by electropharesis on a 1.4% agarose gel and then transferred to Zetabind (Biorad) membrane according to standard procedures. The 3bp deletion of eI507 can be revealed by a very convenient polyacrylamide gel electrophoresis procedure. When the PCR products generated by the above-mentioned 10i-5 and 10i-3 primers SUST~T TE SKEET.

W~O 91/1073a PC'f/CA91/00009 ~ 11 ~d~ 7 97 ,~~,9 ~J~_ø. :.
are applied to an 5~ polyacrylamide gel, sleetrophoresed f~r 3 hrs at 20V/cm in a 90mM Tris-borate buffer (pFi 8.3j, DNA fragments of a different mobility are clearly detectable for individuals without the 3 by deletion, heterozygous or homozygous for the deletion.
As already explained with respect to Figure 20, the PCR amplified genomic DNA can be subjected to gel electrophoresis to identify the 3 by deletion. As shown in Figure 20, in the four lanes the first lane is a control with a normal/oF508 deletion. The next lane is the father with a normal/0I507 deletion. The third lane is the mother with a normal/oF508 deletion and the fourth lane is the child with a eF508/aI50? deletion. The homoduplexes show up as solid bands across the base of each lane. In lanes 2 and 3, the two heteroduplexes show up very clealy as two spaced apart bands. In lane 2, the father's bI507 mutation shows up very clearly, whereas in the fourth lane, the child with the adjacent 507, 508 mutations, there is no distinguishable heteroduplexes.
Hence the showing is at the homoduplex line. Since the father in lane 2 and the mother in lane 3 show heteroduplex banding and the child does not, indicates either the child is normal or is a patient. This can be father checked i~ needed, such as in embryoic analysis by mixing the 507 and 508 probes to determine the presence of the dI507 and 0F508 mutations.
Simihar alteration in gel mobility for heteraduplexes formed during PCR has also been reported for experimental systems where small deletions are involved (Nagamine et al sux~ra). These mobility shifts may be used in general as the basis for the non-radioactive genetic careening tests.
~sr~, CF' 8~$NI3llf~ hROaR.nMB
It is appreciated that approximately 1% of the carriers can be detected using the specific 0I507 probes of this particular embodiment of the invention. Thus, if an individual tested is not a carrier using the aI5o7 WO 91/10734 P~f/CA91100009 c'~ r.. a ;;
~,4~ ~~:~m . 98 probes, their carrier status can not be excluded, they may carry same other mutation, such as the oF508 as previously noted. However, if both the individual and the spouse of the individual tested are a carrier for the eI507 mutation, it. can be stated with certainty that they are an at risk couple. The sequence of the gene as disclased herein is an essential prerequisite for the determination of the other mutations.
Prenatal diagnosis is a logical extension of carrier screening. A couple can be identified as at risk for having a cystic fibrosis child in one of two ways: if they already have a cystic fibrosis child, they are both, by definition, obligate carriers of the defective CF'TR
gene, and each subsecguent child has a 25% chance of being affected with cystic fibrosis. A major advantage of the present invention eliminates the need for family pedigree analysis, whereas, according to this invention, a gene mutation screening program as outlined above or other similar method can be used to identify a genetic mutation that leads to a protein with altered function. This is not dependent on prior ascertainment of the family through an affected child. Fetal DNA samples, for example, can be obtained, as previously mentioned, from amniotic fluid cells and chorionic villi specimens.
Amplification by standard PCR techniques can then be performed on this template DNA.
If b~th parents are shown to be carriers with the 0I507 deletion., the interpretation of the results would be the following. If there is hybridization of the fetal DNA to the normal probe, the fetus will not be affected with. cystic fibrosis, although it may be a CF carrier (50% probability for each fetus of an at risk couple). If the fetal DNA hybridizes only to the aI507 deletion probe arid not to the normal probe, the fetus will be affected with cystic fibrosis.
It is appreciated, that for this and other mutations in the CF gene, a range of different specific procedures 1V0 91 /10734 PC~'/CA91 /00009 99 n'v'j''L''i.:B
can be used to provide a complete diagnosis for all potential CF carriers or patients. A complete description of these procedures is later described.
The invention therefore provides a method and kit for determining if a subject is a CF carrier or CF
patient. In summary, the screening method comprises the steps of:
providing a biological sample of the subject to be screened; and providing an assay for detecting in the biological sample, the presence of at least a member from the group consisting of a 507 mutant CF gene, 507 mutant CF gene products and mixtures thereof.
The method may be further characterised by including at least one more nucleotide probe which is a different DNA sequence fragment of, for example, the DNA of Figure 1, o~' a different DNA sequence fragment of human chromosome 7 and.located to either side of the DNA
sequence of Figure 1. In this respect, the DNA fragments of the intron portions of Figure .2 are useful in further confirming the presence of the mutation. Unique aspects of the introns at the axon boundaries may be relied upon in screening procedures to further confirm the presence of the mutation at the T507 position or othe mutant positions.
A kit, according to an embodiment of the invention, suitable for use in the screening technique and for assaying for the presence of the mutant CF gene by an immunoassay comprises:
(a) an antibody which specifically binds to a gene product of the mutant CF gene having a mutation at one of the positions c~f 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092;
(b) reagent means for detecting the binding of the antibody to the gene product; and (c) the antibody and reagent means each being present in amounts effective to perform the immunoassay.
~J ~'~ I'T '~ S E E'~

~~ 91/10734 PCT/CA91/00009 c ~~ 't h~ kit for assaying for the presence for the mutant CF gene may also be provided by hybridization technidues.
The kit comprises:
(a) an oligonucleotide probe which specifically binds to the mutant CF gene having a mutation at one of the positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092;
reagent means for detecting the hybridization of the oligonucleotide probe to the mutant CF gene; and (c) the probe and reagent means sash being present in amounts effective to perform the hybridization assay.
'---_ ~~s 3~o~T ~~rT~
As mentioned, antibodies t~ epitopes within the mutant CFTR protein at positions 85, 148, x.78, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092 are raised to provide extensive information on the chara~cter.istics of the mutant protein and othex° valuable information which includes:
1. The antibodies can be used to provide another 2o technicjue in detecting any of the other CF mutations which result in the synthesis of a protein with an altered size.
2~ Antibodies to distinct domains of the mutant protein can be used to determine the topological _ arrangement of the protein in the cell membrane.
This provides informa~tian on segments of the protein which are accessible to externally added modulating agents for purposes of drug therapy:
3. ~'he structure-function relationships of 3o portions of the protein can be examined using specific antibodies. For example, it is possible to introduce into cells antibodies recognizing each of the charged cytoplasmic loops which join the transmembrane sec;uences as well as portions of the nucleotide binding folds and the R-domain, The influence o! these antibodies on functional Parameters of the protein provide insight into cell ~i 101 ~~'~~~C'~.w regulatory mechanisms and potentially suggest means of modulating the activity of the defective protein in a CF patient.
4. Antibodies with the appropriate avidity also enable immunoprecipitation and immuno-affinity purification of the protein. Immunoprecipitation will facilitate characterization of synthesis and post translational modificatian including ATP
binding and pt~osphorylation. Purification will be required for studies of protein structure and for reconstitution of its function, as well as protein based. therapy.
In order to prepare the antibodies, fusion proteins contaaning~defined portions of anyone of the mutant CFTR
polypeptides can be synthesized in bacteria by expression of corresponding mutant DNA sequence in a suitable cloning vehicle. Smaller peptide may be synthesized chemically. The fusian proteins can be purified, for example, by affinity chromatography on gluta~thione-agarose and the peptides coupled to a carrier protein (he~aocyanin), mixed with Freund~s adjuvant ahd injected into rabbits. JFollowing booster injections at bi-weekly intervals, the rabbits are bled and sera isolated. The developed polyclonal antibodies in the sera gay then be combined with the fusion proteins. Immunablots are then firmed by staining with, for e~cample, alkaline-phosphatase conjugated second antibody in accordance with the procedure of Make et al, ~nal,,~Hiochem. 136:175 X1984).
Thus, it is possible to raise polyclonal antibodies specific for both fusion proteins containing portions of the mutant CFTR protein and peptides corresponding to short segments of its sequence. Similarly, mice can be injected with KLH conjugates of peptides to initiate the production of anonoclonal antibodies to corresponding seglnentB ~f Alutant C.~''TR prCtein.

lVO 91/10734 PCT/CA~1/00009 ~~ ~i ~( !~ .~s for the generation of monoclonal antibodies, immunogens for the raising of monoclonal antibodies (mAbs) to the mutant CFTR protein are bacterial fusion proteins [smith et al, 67:31 (1986)j containing portions of the cF~t polypeptide or synthetic peptides corresponding to short (12 to 25 amino acids in length) segments of the mutant sequence. The essential methodology is that of Kohler arid i~Iilsteln [N~tuj~ 256:
495 (1975)j.
Balb/c mace are immunized by intraperitoneal injection with 500 ~cg of pure fusion protein or synthetic peptide in incomplete Freund~s adjuvant. A second injection is given after 14 days, a third after 21 days and a fourth after 2s days. Individual animals so immunized are sacrificed one, two and four weeks following the final injection. Spleens are removed, their cells dissociated, collected and fused with Sp2/0-Agl4 myeloma cells according to Defter et al, Somati,g ~'~~1 Genet ~a 3:231 (197'7). The fusion mixture is distributed in culture ~aedium selective for the pr~pagation of fused cells which are grown until they are about 25~ confluent. At this time, culture supernatants are tested for the presence of antibodies reacting with a particular CFTR antigen. An alkaline phosphatase labelled anti-mouse second antibody is then used for detection of positives. Cells from positive culture wells acre than expanded in culture, their supernatants collected for further testing and the cells stored deep fx°ozen in crycaprotectant-containing medium. To obtain large quantities of a mAb, producer cells are injected into the peritoneum at 5 x 106 cells par animal, and ascites fluid is obtained. Purification is by chromotography on Protein G- or Protein A-agarose according to Ey et al, Im_munochem;~i,-~. 15:429 (1977), ' Reactivity of these mAbs with the mutant CFTR
protein can be confirmed by polyacrylamide gel electrophoresis of membranes isolated from epithelial '0~0 91 / 10734 ~'CT/Cr~91 /00009 103 ~~a T~ a c7' ':i cells in which it is expressed and immunoblotted [Towbin et al, P~~g. t3atl. Acad. Sci. USA 76:4350 (1979) ] .
In addition to the use of monoclonal antibodies specific for the particular mutant domain of the CFTR
protein to probe their individual functions, other mAbs, which can distinguish between the normal and mutant forms of CFTR protein, are used to detect the mutant protein in epithelial cell samples obtained from patients, such as nasal mucosa biopsy ~~brushings" [ R. De-Lough and J.
Rutland, 3. C7,in.~at ol. 42, 613 (1989) ] or stein biopsy specimens containing sweat glands.
Antibodies capable of this distinction are obtained by differentially screening hybridomas from paired sets of mice immunized with a peptide containing, for example, the isoleucirae at amino acid position 507 (e. g.
GTIKENI,~FGVSY) or a peptide which is identical except for the: absence of 1507 (GTIKENIFGVSY). mAbs capable of recognizing the other mutant forms of CFTR protein present in patients in addition or instead of 1507 2o deletion are obtained using similar monoclonal antibody production strategies.
antibodies to normal and CF versions of CFTR protein and of segments thereof are used in diagnostically immunocytochemical and immunofluorescence light microscopy and immunoelectron microscopy to demonstrate the tissue, cellular and subce11u1ar distribution of CFTR
within the organs of CF patients, carriers and non-CF
individuals.
l~ntibodies are used to therapeutically modulate by 3o promoting the activity of the CFTR protein in CF patients and in cells of CF patients. Possible modes of such modulation might involve stimulation due to cross-linking of CFTR protein molecules with multivalent antibodies in analogy with stimulation of some cell surface membrane receptors, such as the insulin receptor [O~Brien et al, EurQ. ~o~, B,~o,).. Organ. J. 6:4003 (1987) ], epidermal growth factor receptor [Schreiber et a1, J. Biol. Chem.

~d ~ ~; ix ~_ 25s:s46 (1983), and T-cell receptor-associated molecules such as CD4 (veillette et al ~latt~tre, 338 0 257 (1989) ] , Antibodies are used to direct the delivery of therapeutic agents to the cells which, eXpress defective cFTR protein in cF. For this purpose, the antibodies are incorporated into a vehicle such a;s a liposome [Matthay et al, Cancer Res. 46:4904 (a986), which carries the therapeutic agent such as a drug or the normal gene.
~.5 RfZP ANA~XSIS
to DNA diagnosis is currently being used to assess whether a fetus will be barn with cystic fibrosis, but historically this has only been done after a particular set of parents has already had one cystic fibrosis child which identifies them as obligate carriers, However, in combination with carrier detection as outlined above, DNA
'diagnosis fox all pregnancies of carrier couples will be pe~sible. If the parents have already had a cystic fibrosis child, an extended haplotype analysis can be done an the fetus and thus the percentage of false positive or false negative will be greatly reduced. If the parents have not already had an affected child and the DNA diagnosis on.the fetus is being performed on the basis of carrier detection, haplotype analysis can still be performed.
Although it has been thought for many years that there is a great deal of clinical heterogen~aty in the cystic fibrosis disease, it is now emerging that there are two general categories, called pancreatic sufficiency (CF-PS) and pancreatic insufficiency (CF-PI). If the mutations related to these disease categories are well characterized, one can associate a particular mutation with a clinical phenotype of the disease. This allows changes in the treatment of ~ach patient. Thus the nature of the mutation will to a certain extent predict the prognosis of the patient and indicate a specific treatment.

W~ 91110734 P~'/~A9111)0009 l05 ~o~~c~ ~xo~oo~ o~ cx~~TC~ ~~~~os~~
The postulate that CFTR may regulate the activity of ion channels, particularly the outwardly rectifying C1 channel implicated as the functional defect in CF, can be tested by the injection and translation of full length in vitro transcribed CFTR mRNA in Xenopus oocytes. The ensuing changes in ion currents across the oocyte membrane can be measured as the potential is clamped at a fixed value. CFTR may regulate endogenous oocyte channels or it may be necessary to also introduce epithelial cell RNA to direct the translation of channel proteins. LTse of mRNA coding for normal and for mutant CFTR, as provided by this invention, makes these experiments possible.
i5 ~ther modes of expression in heterologous cell system also facilitate dissection of structure-function relationships. The complete CFTR DNA sequence ligated into a plasmid expression vector is used to transfect cells so that its influence on ion transport can be assessed. Plasmid expression vectors containing part of the normal CFTR sequence along with portions of modified sequence at selected sites can be used in vitro mutagenesis experiments performed in order to identify those portions of the CFTR protein which are crucial for regulatory function.
~s.,& EXPItE~~IOP1 !~~ 'fRE ~ItJTA..~'9' DNFv 8 nrgv~rng The mutant DNA sequence can be manipulated in studies to understand the expression of the gene and its product, and, to achieve production of large quantities of the protein for functional analysis, antibody production, and patient therapy. The changes in the sequence may or may not alter the expression pattern in terms of relative quantities, tissue-specificity and functional properties. The partial or full-length cDNA
sequences, which encode for the subject protein, unmodified or modified, may be ligated to bacterial expression vectoxs such as the pRTT (Nilsson et al. EMB_O

wo ~mo7~ pcri~A~lioooo~
~ ~'~ ~ !.~ C~ 106 ,~, 4: 1075-1080 (1985)), pGEX (Smith and Johnson, 67: 31-40 (1988)) or pATIi (8pindler et al. J. V x-01. 49:
132-141 (1984)) plasmids which can be introduced into ~.
coli cells for production of the corresponding proteins which may be isolated in accordance with the previously discussed protein purification procedures. The DNA
sequence can also be transferred from its existing context to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal virus, yeast to artificial chromosomes (YAC)(Burke et al. Saie re 236;
8os-812, (1987)), somatic cells, and ether simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, ,~ fence 244: 1313~-1317 (1989), invertebrates, plants (Gasser and Fraley, a a 244:
1293 (1989), and pigs (Pursel et al. ~_ience 244: 1281-128s (19s9)).
. For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV) 40, promoter in the pSV2 vector [Mulligan and Berg, DoE, Nat, Acad ~~t yran, ?8:2072-2076 (1981)] and introduced into cells, such as monkey CCS-1 cells [Gluzanan, C~1~,, 23:17x-182 (1981)J, t4 achieve transient or long-term expression. The stable integration of the chimeric gene construct may be 2S maintained in mammalian sells by biochemical selection, such as neomycin [Southern and Berg, J. Mol, Aooln.
Genet. 1:327-341 (1982):] and mycophoenolic acid (Mulligan and Berg, DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence-alteration via single-stranded bacteriophage intermediate or with the use of specific ol'igonucleotides in combination with PCR.

'WO 91/10734 PC.T/CA91/OO~Dlf9 107 ~~J~C~~~/~ ~l.x The cDNA sequence (or portions derived from it), or a mini gene (a cDNA with an intron and its own promoter) is introduced into eukaryotic expression vectors by conventional techniques. These vectors are designed to permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. Vectors containing the promoter and enhancer regions of the simian virus ~o (sv)4o or long terminal repeat (LTR) of. the Rous Sarcoma virus and polyadenylation and splicing signal from SV 40 are readily available [Mulligan et al Proc. Natl Acad Sci. LISA 78:1078-2076, (1981); GOrmari et al ProC Nail ACad. Sci tiaA 79: 6777-6781 (1982)j. Alternatively, the CFTR endogenous promoter may be used. The level of expression of the cDNA can be manipulated with this type of vector, either by using promaters tPaat have different activities (for ea~ample, the baculovirus pAC373 can express cDNAs at high levels in ;~,. ~runaincells [M.
2o D. Summers and G. E. Smith in, Genetically Altered viruses and the Environment (B. Fields, et al, eds.) vol.
22 no 3i9-328, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, 1985] or by usang vectors that contain promoters amenable to modulation, for example the glucocorticoid-responsive promoter from the mouse mammary tumor virus [I~e et al, P~ature 294:228 (1982)J. The expression of the cDNA can be monitored in the recipient cells 24 to 72 hours after introduction (transient expression).
In addition, some vectors contain selectable markers [such as the ,gp~, [Mulligan et Berg supra] or neo [Southern and Berg ~. Mol. Aptaln Gp~p~ ~,:327-3d1 (1982)]
bacterial genes that permit isolation of cells, by chemical selection, that have stable, long term expression of the vectors (and therefore the cDNA) in the recipient cell. The vectors can be maintained in the cells as episomal, freely.replicating entities by using v 1 108 ~~ ~~~~i~?
regulatory elements of viruses such as papilloma [Server et al Mol. Cell lBiol~ 8:486 (1981)] or Epstein-Barr (Sugden et al MOI. Cei_t yiol 5:410 (1985)].
Alternatively, one can also produce cell lines that have integrated the vector into genomic DNA. Both of these types of cell lines produce the gene product on a continuous basis. One can also produce cell lines that have amplified the number of copies of the vector (and therefore of the cDNA as well) to create cell lines that can produce high levels of the gene product [Alt et al.
J~ Bio1 them 253: 1357 (1978)].
The transfer of DNA into eukaryotic, in particular human or other mammalian cells is now a conventional technique. The vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate [Graham and vender Eb, Virolocrv 52:466 (1973) or strontium phosphate [Brash et al Mol. Cell Bio, 7:2013 (1987)], electroporation [Neumann et al EMBO J 1:841 (1982)J, lipofect~.on [Felgner ~r-i rmn et al Pros Nato Aged 84.7413 (1987)J, DEAF
dextran [McCuthan et al ~. Nat_'! Can_ce~r Tns~_ 41;351 1968)J, microinjection [Mueller et al Cellq15:579 1978)], protoplast fusion [Schafner, Proc Natl AGE Sci tten 72:2163) or pellet guns [Klein et al, Na~u~g 327: 70 (1987)]. Alternatively, the cDNA can be introduced by infection with virus vectors: Systems are developed that use, for example, retroviruses [Bernstein et al. ~en~t~,,~
F~ n ina 7: 235, (1985)), adenoviruses [Ahmad et al T~
,Vi~rol 5'1:267 (1986)) or Herpes virus [Spaete et al Cell 30:29 (1982)].
These eukaryotic expression systems can be used for many studies of the mutant CF gene and the mutant CFTR
product,.such as at protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1,077 and 1092.
These include, for example: (1) determination that the gene is properly expressed and that all post-translational modifications necessary for full biological !v0 91110734 PCT/CA91/00009 109 ~3~~~~~1~~.~, activity have been properly completed (2) identify regulatory elements located in the 5o region of the CF
gene and their role in the tissue~ or temporal-regulation of the expression of the CF gene (3) production of large amounts of the normal protein for isolation and purification (4) to use cells expressing the CFTR protein as an assay system for antibodies generated against the cFTR protein or an assay system to test the effectiveness of drugs, (5) study the function of the normal complete to protein, specific portions of the protein, or of naturally occurring or artificially produced mutant proteins. Naturally occurring mutant proteins exist in patients with CF while artificially produced mutant protein can be designed by site directed sequence 1S alterations. These latter studies can probe the function of any desired amino acid residue in the protein by mutating the nucleotides coding far that amino acid.
Using the above techniques, the expression vectors containing the mutant CF gene sequence or fragments 2o thereof can be introduced into human cells, mammalian cells from other species ar non-mammalian sells as desired. The choice of cell is determined by the purpose of the treatment. For example, one can use monkey COS
cells ~Gluzman, Cell 23:175 (1981),, that produce high 25 levels of the SV4o T antigen and-permit the replication of vectors containing the SV40 origin of replication, can be used to show that the vector can express the protein product, since function is not required. Similar treatment could be performed with Chinese hamster ovary 30 (CH~) or moues NZH 3T3 fibroblasts or with human fibroblasts or lymphoblasts.
The recombinant cloning vector, according to this invention, then comprises the selected DNA of the DNA
sequences of this invention for expression in a suitable 35 hash The DNA is operatively linked in the vector to an expression contral.sequence in the recombinant DNA
molecule so that normal CFTR polypeptide can be WO 91!10734 P(.°TlCA91/00009 ~a~~ .~~,'~C~ ~ mo expressed. The expression control sequence may be selected from the group consisting of sequences 'that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof. The expression control sequence may be specifically selected from the group consisting of the ~ system, the t~ system, the system, the ~c system, major operator and promoter regions of phage lambda, the control region of id coat protein, the early 1.o and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculavirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof, as The host cell, which may be transfected with the vector of this invention, may be selected from the group consisting of ~, coli, pseudomonas, Saci_Wuc sub 'l y ~~ci pus s~earothe moph9lus or other bacili; other bacteria; yeast; fungi; insect; mouse or other animal;
20 or plant hosts; or human tissue cells.
It is appreciated that for the mutant DNA sequence similar systems are employed to express and produce the mutant product.
z!ROT~zN ~mN~~~orr ~orrszn~~~r_Tnu~
25 To study the function of the mutant CFTR protein, it is preferable to use epithelial cells as recipients, since proper functional expression may require the presence of other pathways or gene products that are only expressed in such cells. Cells that can be used include, 30 for example, human epithelial cell lines such as T84 (ATCC ,~CRL 248) or FANC-1 (ATCC ,~ CLL 1459), or the T43 immortalised CF nasal epithelium cell line [Jettan et al, ,~.ence (1989) and primary [Yanhoskes et al. Ann. Rev.
Res~a. Dis 132: 1281 (1985)) or transformed [Scholte et 35 al. Exc. Cell. Res 182: 559(1989)] human nasal polyp or airways cells, pancreatic cells [Harris and Coleman ~, Cell. Sci 87: 695 (298?),, or sweat gland cells [Collie iy0 91/10734 1'CT/CA91/00009 111 ~~~'~~~u~(a:y_ et al. ,fin Vi~ro 21s 597 (1985)] derived from normal or CF
subjects. The CF cells can be used to test far the functional activity of mutant CF genes. current functional assays available include the study of the movement of anions (C1 or I) across cell membranes as a function of stimulation of cells by agents that raise intracellular AMP levels and activate chloride channels [Stutto et al. g~,Qc. Nat. Aced. Sci. U. S. .A. 82: 6677 (1985)]. Other assays include the measurement of changes to in cellular potentials by patch clamping of whole cells or of isolated membranes [Frizzell et a1. Science 233:
558 (1986), Welsch and Liedtke 322: X67 (1986)]or the study of ion fluxes in epithelial sheets of confluent cells [Widdicombe et al. Proc. Nat. Aced. Sci. 82: 6167 (1985)]. Alternatively, RNA made from the CF gene could be injected into ~,~nogus oocytes. The oocyte will translate RNA into protein and allow its study. As other more specific assays are developed these can also be used in the study of transfected mutant CFTR protein function.
~~pomain-switching~~ experiments between aautant CFTR
and the human multidrug resistance P-glycoprotein can also be performed to further the study of the mutant CFTR
protein. In these experiments, plasmid expression vectors are constructed by routine techniques from fragments of the mutant CFTR sequence and fragments of the sequence of P-glycoprotein ligated together by DNA ligase so that a protein containing the respective portions of these two proteins will be synthesized by a host cell transfected with the plasmid. The latter approach has the advantage 3o that many experimental parameters associated with multidrug resistance can be measured. hence, it is now possible to assess the ability of segments of mutant CFT~t to influence these parameters.
These studies of the influence of mutant CFTR on ion transport will serve to bring the field of epithelial transport into the molecular arena.

'WO 91/10734 PCT/CA91/00009 ~~~~I~ y ~ 112 _',~L'P~IaB
It is understood that the major aim of the various biochemical studies using the compositions of this invention is the development of therapies to circumvent or overcome the CF defect, using both the pharmacological and the "gene-therapy" approaches.
In the pharmacological appraach, drugs which circumvent or overcome the CF defect are sought.
Initially, compounds may be tested essentially at random, and screening systems are required to discriminate among many candidate compounds. This invention provides host cell systems, expressing various of the mutant CF genes, which are particularly well suited for use as first level screening systems. Preferably, a cell culture system using mammalian cells (most preferably human cells) transfected with an expression vector comprising a DNA
sequence coding for CFTR protein captaining a CF-generating mutation, for example the 1507 deletion, is used in the screening process. Candidate drugs are 20, tested by incubating the cells in the presence of the candidate drug and measuring those cellular functions dependent on CFTR, especially by measuring ion currents ~rhere the transmembrane potential is clamped at a fixed value. To accommodate tha large number of assays, however, more convenient assays are based, for example, on the use of ion-sensitive fluorescent dyes. To detect changes in C1''on concentration SPA or its analogues are useful.
Altera~atively, a cell-free system could be used.
Purified CFTR could be reconstituted into articifial membranes and drugs oould be screened in a cell-free assay [A1-Aqwatt, ,S,~ience, (1989)x.
At the second level, animal testing is required. It is possible to develop a model of CF by interfering with the normal expression of the counterpart of the CF gene in an animal such~as the moue. The "knock-out" of this gene by introducing a mutant form of it into the germ WO 91/10734 1'C'f/CA91/00009 l~~ ~, L ~ ~ ~ ~i line of animals will provide a strain of animals with cF-like syndromes. This enables testing of drugs which showed a promise in the first level cell-based screen.
As further knowledge is gained about the nature of the protein and its function, it will be possible to predict structures of proteins or other compounds that interact with the CFTR protein. That in turn will allow for certain predictions to be made about potential drugs that will interact with this protein and have some effect ~o on the treatment of the patients. Ultimately such drugs may be designed and synthesized chemically on the basis of structures predicted to be required to interact with domains of CFTR. This approach is reviewed in Capsey and Delvatte, a Stockton press, ldew York, x.988. These potential drugs must also be tested in the screening system.
PI~oT~Ild REPr:a~~at~am ~ror~ru!naw Treatment of CF can be performed by replacing the defective gratein with normal protein, by modulating the 2o function of the defective protein or by modifying another step in the pathway in which CFTR participates in order to correct the physiological abnormality.
To be able to replace the defective protein with the normal version, one must have reasonably large amounts of pure CFTR protein. Pure protein can be obtained as described earlier dram cultured cell systems. Delivery of the protein to the affected airways tissue will require its packaging in lipid-containing vesicles that facilitate the incorporation a! the protein into the cell membrane. It may also be feasible to use vehicles that incorporate proteins such as surfactant protein, such as SAP(Val) or sAP(Phe) that performs this function naturally, at least for lung alveolar cells. (PCT patent Application WO/8803~.70, Whitsett et a1, May 7, 1888 and PCT patent Application W089/04327, Benson et al, May 18, 1989). The CFTR-containing vesicles are introduced into WCa 91/10734 I'C~'/C~91/00009 ~~~~~~~.i 114 the airways by inhalation or irrigation, techniques that are currently used in CF treatment (Boat et al, supxa).
6.3.2 DISUGf '~'~I~PX
Modulation of CFTR function can be accomplished by the use of therapeutic agents (drugs). These can be identified by random approaches using a screening program in which their effectiveness in modulating the defective CFTR protein is monitored ,~ vitro. Screening programs can use cultured cell systems in which the defective CFTR
protein is expressed. Alternatively, drugs can be designed to modulate CFTR activity from knowledge of the structure and function correlations of CFTR protein and from knowledge of the specific defect in the CFTR mutant protein (Capsey and Dalvatte, ). It is possible that the mutant CFTR protein will require a different drug for specific modulation. It, will then be necessary to identify the specific mutations) in each CF patient before initiating drug therapy.
Drugs can be designed to interact with different aspects ~f CFTR protein structure or function. For example, a drug (or antibody) can bind to a structural fold of the protein to correct a defective structure.
Alternatively, a drug might bind to a specific functional residue and increase its affinity for a substrate or cofactor. Since it is known that members of the class of proteins to which CFTR has structural homology can interact, bind and transport a variety of drugs, it is reasonable to expect that drug-related therapies may be effective in treatment of CF.
A third mechanism for enhancing the activity of an effective drug would be to modulate the production or the stability of CFTR inside the cell. This increase in the amount of CFTR could compensate for its defective function.
Drug therapy can also be used to compensate for the defective CFTR function by interactions with other components of the physiological or biochemical pathway w~ 9aiao~~ pcricA~noooo9 '~~~t~.l~.
necessary for the expression of the CFTIt function. These 'interactions can lead to increases or decreases in the activity of these ancillary proteins. The methods for the identification of these drugs would be similar to those described above for GFTR-related drugs.
2n other genetic disorders, it has been possible to correct for the consequences of altered or missing normal functions by use of dietary modifications. This has taken the form of removal of metabolites, as in the case to of phenylketonuria, where phen~lalanine is removed from the diet i.n the first five years of life to prevent mental retardation, or by the addition of larger amounts of metabolites to the diet, as in the case of adenosine deaminase deficiency where the functional correction of the activity of the enzyme can be produced by the addition of the enzyme to the diet. Thus, once the details of the CFTR function have been elucidated and the ~aWic defect in CF has been defined, therapy may be achieved by dietary manipulations.
2~ The second potential therapeutic approach is so-called "gene-therapy" in which normal copies cf the CF
gene errs introduced in to patients so as to successfully code far normal protein in the key epithelial cells of affected tissues. It is most crucial to attempt to achieve this with the airway epithelial cells of the respiratory tract. Ths CF gene is delivered to these cells fn form in which it can betaken up and code for sufficient protein to provide regulatory function. As a result, "the patient s quality and length of life will be greatly extended. tTltimately, of course, the aim is to deliver the gene to all affected tissues.
One appraach to therapy of CF is to insert a normal version of the CF gene into the airway epithelium of affected patients. It is important to note that the respiratory system is the primary cause of mordibity and mortality in CF; while pancreatic disease is a major WO 91/10734 PCT1CA91/(~09 15.6 feature, it is relatively well treated today with enzyme supplementation. Thus, somatic cell gene therapy (for a review, see T. Friedmann, a ee 244:1275 (1989)) targeting the airway would alleviate the most severe problems associated with CF.
A. Retroy~ra7 Vectors. Retroviruses have been considered the preferred vector for experiments in somatic gene therapy, with a high efficiency of infection and stable integration and expression [orkin et al Pr~a.
20 Med. Genet 7:130, (1988}J. A possible drawback is that cell division is necessary for retroviral integration, so that the targeted cells in the airway may have to be nudged into the call cycle prior to retroviral infection, perhaps by chemical means. The full length CF gene cDNA
can be cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LRT
(Long teruinal repeat). Exgression of levels of the normal protein as low as 1o% of the endogenous mutant protein in CF patients would be expected to be 2o beneficial, since this is a recessive disease. Delivery of the virus could be accomplished by aerosol or instillation into the trachea.
B. other ~ira~ vo.a~,.ao Other delivery systems which can be utilized include adeno-associated virus [AAV, McLaughlin et al, J. Viroi 62:1963 (1988)], vaccinia, virus [doss st al Annu. ev '~ > >nol, 5:3~s, 1987)1, bovine gegil1oma virus [Rasmussen et al, J~gt ode ], 139:642 (1987)] or ~e:aber of the hergesv3rus group such as Epstein-Barr virus (Margolskee et al ~tol.
3o Cel1.B~o~ 8:2937 (1988),. Though much would need to be learned about their basic biology, the idea of using a viral vector with natural trogism for the respiratory track (e. g. respiratory syncytial virus, echovirus, Coxsackie virus, etc.) is possible.
C. Non_-vira~ Gene Tran~fp.-, ether methods of inserting the CF gene into respiratory epithelium may also be productive; many of these are lower efficiency WO 91/1074 ~'~Ci°/CA9IlOOOU9 11? ~~~ 3 and would potentially require infection ,~ vitro, selection of tr~nsfectants, and reimplantation. Thus would include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. A particularly attractive idea is the use of liposome, which might be possible to carry out ,~ vivo (Ostro, ~ioo~ spores, Marcel-Dekker, 198?]. Synthetic cationic lipids such as DOTrIA
[Felger et al pros N~. t1 AC~~ ~ni TTc~ 84:?413 (198?) ]
may increase the efficiency and ease of carrying out this approach.
s . ~, c~ Artzr~ ~oaELa The creation of a mouse or other animal model for CF
will be crucial to understanding the disease and for testing of possible therapies (for general review of creating animal models, see Erickson, Am. ,? Hum G n t 43:582 (1988)]. Currently no animal model of the CF
exists, The evolutionary conservation of the CF gene (as demonstrated by the cross-species hybridization blots for E4.3 and H1.6), as is Shawn in Figure 4, indicate that an orthologous gene exists in the mouse (hereafter to be denoted mCF, and its corresponding protein as mCFTR), and this wall be possible to clon~ in mouse genomic and cDNA
libraries using the human CF gene probes. It is expected that the generation of a specific mutation in the mouse gene analogous to the T50? mutation will be most optimum to reproduce the phenotype, though complete inactivation of the mCFTR gene will also be a useful mutant to generate.
'A~ d~utagL, esis Inactivation of the mCF gene can be achieved by chemical [e. g. Johnson et al pros, Hats, Acad, Sci. USA 78:3138 (1981)] or 7(-ray mutagenesis [Popp et al J. Mol. Bln~_ 12?:141 (19?9)] of mouse gametes, followed by fertilization. Offspring heterozygous fox inactivation of mCFTR can then be identified by Southern blotting to demonstrate loss of one allele by dosage, or failure to inherit one parental allele if an RFLP marker is being assessed. This approach has previously been WO 91/10734 PCg'/~A91/00009 ~~~~~~~ C
ma successfully used to identigy mouse mutants for a-globin [~hltney et al ~~OC Nat3 $r~a~ Cyi U~"A 77 v a.o8'~
(1980)), phenylalanine hydroxylase [McDonald et al Pediatr Res 23:63 (1988)), and carbonic anhydrase II
[Lewis et al pr c. Natl Acad ~r~~TCA 85~1c362, (198g)), B. TransA mutant version og CFTR or mouse CFTR can be inserted into the mouse germ line using now standard techniques of oocyte injection [Camper, a ds in Genetics (1988)l; alternatively, if it is desirable to 1o inactivate or replmce the endogenous mCF gene, the homologous recombination system using embryonic stem (ES) cells [Capecchi, 'ence 244:1288 (1989)] may be applied.
1. oocvte _rni ion placing one or more copies of the normal or mutant mCF gene at a random location in the mouse germline can be accomplished by microinjection of the pronucleus of a just-fertilized mouse oocyte, followed by reimplantation into a pseudo-pregnant foster mother. The liveborn mice can then be screened for integrants using mnalysis of trail DIdA for the presence of 2o human CF' gene sequences. The same protocol cmn be used to insert a mutmnt mCF gene. To generate m mouse model, one would want to plmce this trmnsgene in m mouse background where the endogenous mCF gene has been inactivated, either by mutagenesi.s (see above ) or by homologous recombination (see,below). The transgene can be eithers m) a complete genomic .sequence, though the size of this (mbout 250 kb) would require that it be injected as a yeast artificial chromosome or a~ chromosome frmgment; b) m cDNA with either the natural promoter or a heterologous promoter; c) a °~minigene" contmining all of the coding region and various other elements such ms introns, promoter, and 3° flanking elements found to be necessmry for optimum expression.
2. Retrovira~ Tnt~er ton o This mlternative involves inserting the CFTR or mCF.gene into a retroviral 'vector and directly infecting mouse embroyos at early stages of development generating a W('D 91 /10734 PtT/CA91 /00009 ~ l9 ~ ~e~ ~,~ ~ ~ /_~
chimera [Soriano et al Gel1 46:~.g (1986)]. At least some of these will lead to germline transmission.
3. ~$ CellS and Hnmnlnnnnc yo.. 1.t ~~ The embryonic stem cell approach (Capecchi, and s Capecchi, Trends Genet 5:70 (1989)] allows the possibility of performing gene transfer and then screening the resulting totipotent cells to identify the rare homologous recombination events. c7nce identified, these can be used to generate chimeras by injection of 1o mouse blastocysts, and a proportion oz the resulting mice will show germline transmission from the recombinant line. There are several ways this could be useful in the generation of a mouse model for CF:
a) Inactivation of the mCF gene can be conveniently 15 accomplished by designing a DNA fragment which contains sequences from a mCFTR axon flanking a selectable marker such as neo. Homologous recombination will lead to insertion of the neo sequences in the middle of an axon, inactivating mCFTR. The homologous recombination events 20 (usually about 1 in 1000) can be recognized from the heterologous ones by DNA analysis of individual clones [usually using PCR, Kim et al ~cleic Acids Res 16:8887 (1888), ~oyner et al Nature 338:153 (1989); Zimmer et al sutra, p. 150] ox by using a negative selection against 25 the heterologous events [such as the use of an HSV ,TK
gene at the end of the construct,.followed by the gancyclovir selection, Mansour et al, Nature 336x348 (1988)]. This inactivated mCFTR mouse can then be used to introduce a mutant CF gene or mCF gene containing, for 30 example, the 1507 abnormality or any other desired mutation.
b) It is possible that specific mutants of mCFTR
cDNA be created in one step. For example, one can make a construct containing mCF intron 9 sequences at the 5~
35 end, a selectable n~o gene in the middle, and intro 9 +
axon 10 (containing the mouse version of the I507 mutation) at the 3~ end. A homologous recombination WO 91/10734 Pt.'T/CA91/00009 ~~~~~c~x'' ~ 120 event would lead to the insertion of the nip gene in intron 9 and the replacement of axon 10 with the mutant version.
c) zf the presence of the selectable neo marker in s the intron altered expresson of the mCF gene, it would be possible to excise it in a second hoaaologous recombination step.
d) zt is also possible to create anutations in the mouse germline by injecting oligonucleotides containing 1o the mutation of interest and screening the resulting cells by pCR.
This embodiment of the invention has considered primarily a mouse model for cystic fibrosis. Figure 4 shows cross-species hybridization not only to mouse DNA, but also to bovine, hamster and chicken DNA. Thus, it is contemplated that an orthologous gene will exist in many other species also. Tt is thus contemplated that it will be possible to generate other animal models using similar technology.
20 Although preferred embodiments of the invention have been described herein in detail, it will be understand by those skilled in the art that variatione~ may be made thereto without departing from the spirit of the invention or the scope of the appended claims.

Claims (47)

CLAIMS:

1. A DNA molecule comprising an intronless DNA sequence encoding a mutant CFTR polypeptide having the sequence according to Figure 1 for amino acid residue positions 1 to 1480 and, further characterized by nucleotide sequence variants resulting in deletion or alteration of amino acids of residue positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092.

2. A DNA molecule comprising a DNA sequence encoding a mutant CFTR polypeptide having the sequence according to Figure 1 for DNA sequence positions 1 to 4575 and, further characterized by nucleotide sequence variants resulting in deletion or alteration of DNA at DNA
sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659.

3. A DNA molecule comprising a DNA sequence selected from the group consisting of:
(a) DNA sequences which correspond to a fragment of a selected sequence in claim 1 or 2 including at least 16 nucleotides; and (b) DNA sequences encoding an epitope characteristic of the mutant CFTR protein encoded by at least 18 sequential nucleotides in the selected sequence of claim 1 or 2.

4. The DNA molecule of claim 1 or 2 wherein the DNA
molecule is a cDNA.

5. The DNA molecule of claim 3 wherein the DNA molecule is a cDNA.

6. An RNA molecule comprising an RNA sequence complementary to the DNA sequence recited in claim 3.

7. A nucleic acid probe comprising a nucleotide sequence corresponding to the selected sequence recited in parts (a), or (b) of claim 3.

8. A nucleic acid probe according to claim 7 wherein said sequence comprises AAA GAA AAT ATC TTT GGT GTT, and its complement.

9. A recombinant cloning vector comprising the DNA
molecule of claim 3.

10. The vector of claim 9 wherein said DNA molecule is operatively linked to an expression control sequence in said recombinant DNA molecule so that a mutant CFTR
polypeptide can be expressed, said mutant CFTR
polypeptide being selected from the group of CFTR
polypeptides at mutant positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092, said expression control sequence being selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.

11. The vector of claim 9 wherein said DNA molecule is operatively linked to an expression control sequence in said recombinant DNA molecule so that a mutant CFTR
polypeptide can be expressed, said mutant CFTR
polypeptide being selected from the group of CFTR
polypeptides at mutant DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659, said expression control sequence being selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.

12. The vector of claim 10 or 11 wherein the expression control sequence is selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV4O, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.

13. A host cell transformed with the vector according to claim 9

14. The host cell of claim 13 selected from the group consisting of strains of E.
coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus, or other bacili;
other bacteria; yeast; fungi; insect; mouse or other animal; plant hosts; or human tissue cells.

15. The host of claim 14 wherein said human tissue cells are human epithelial cells.

16. A method for producing a mutant CFTR polypeptide comprising the steps of:
(a) culturing a host cell transfected by the vector of claim 9 in a medium and under conditions favorable for expression of the mutant CFTR polypeptide selected from the group having mutant positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092;
(b) isolating the expressed mutant CFTR polypeptide.

17. A method for producing a mutant CFTR polypeptide comprising the steps of:
(a) culturing a host cell transfected by the vector of claim 9 in a medium and under conditions favorable for expression of the mutant CFTR polypeptide selected from the group having mutant DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659;
(b) isolating the expressed mutant CFTR
polypeptide.

18. A purified mutant CFTR polypeptide substantially free of other human proteins and encoded by the DNA
sequence recited in claim 3.

19. A substantially pure mutant CFTR polypeptide according to claim 18 made by chemical or enzymatic peptide synthesis.

20. A polypeptide coded for by expression of a DNA
sequence recited in accordance with the process of claim 16 or 17.

21. A method for screening in vitro subject to determine if said subject is a CF carrier or a CF patient comprising the steps of:
providing a biological sample from the subject to be screened; and providing an in vitro assay for detecting in the biological sample, the presence of at least a member from the group consisting of a mutant CF gene, a mutant CFTR polypeptide products and mixtures thereof, the mutants being defined by mutations at protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092.

22. A method for screening in vitro a subject to determine if said subject is a CF carrier or a CF patient comprising the steps of:
providing a biological sample from the subject to be screened; and providing an in vitro assay for detecting in the biological sample, the presence of at least a member from the group consisting of a mutant CF gene, a mutant CFTR polypeptide products and mixtures thereof, the mutants being defined.by mutations at DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659.

23. The method of claim 21 or 22 wherein the biological sample includes at least part of the genome of the subject and the assay comprises an hybridization assay.

24. The method of claim 23 wherein the assay further comprises a labelled nucleotide probe according to claim 7.

25. The method of claim 24 wherein said probe comprises the nucleotide sequence of claim 8.

26. The method of claim 21 or 22 wherein the biological sample includes a CFTR polypeptide of the subject and the assay comprises an immunological assay.

27. The method of claim 26 wherein the assay further includes an antibody specific for said mutant CFTR
polypeptide.

28. The method of claim 26 wherein the assay is a radioimmunoassay.

29. The method of claim 27 wherein the antibody is a monoclonal antibody.

30. The method of claim 21 or 22 wherein the biological sample is isolted from a subject which is a human fetus in utero.

31. The method of claim 24 wherein the assay further includes at least one additional nucleotide probe according to claim 7.

32. The method of claim 31, wherein the assay further includes a second nucleotide probe comprising a different DNA sequence fragment of the DNA of Figure 1 or its RNA
homologue or a different DNA sequence fragment of human chromosome 7 and located to either side of the DNA
sequence of Figure 1.

33. In a process for screening a potential CF carrier or patient to indicate the presence of an identified cystic fibrosis mutation in the CF gene, said process including the process steps of:
(a) isolating genomic DNA from said potential CF
carrier or said potential patient;
(b) hybridizing a DNA probe onto said isolated genomic DNA, said DNA probe spanning a mutation in said CF gene wherein said DNA probe is capable of detecting said mutation, said mutation being selected from the group of mutations at protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and (c) treating said genomic DNA to determine presence or absence of said DNA probe and thereby indicating in accordance with a predetermined manner of hybridization, the presence or absence of said cystic fibrosis mutation.

34. In a process for screening a potential CF carrier or patient to indicate the presence of an identified cystic fibrosis mutation in the CF gene, said process including the process steps of:
(a) isolating genomic DNA from said potential CF
carrier or said potential patient;
(b) hybridizing a DNA probe onto said isolated genomic DNA, said DNA probe spanning a mutation in said CF gene wherein said DNA probe is capable of detecting said mutation, said mutation being selected from the group of mutations at DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659.

35. A process for detecting cystic fibrosis carriers of a mutant CF gene wherein said process consists of determining differential mobility of heteroduplex PCR
products in polyacrylamide gels as a result of deletions or alterations in the mutant CF gene at one or more of the protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092.

36. A process for detecting cystic fibrosis carriers of a mutant CF gene wherein said process consists of determining differential mobility of heteroduplex PCR
products in polyacrylamide gels as a result of deletions or alterations in the mutant CF gene at one or more of the DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659.

37. A kit for assaying for the presence of a mutant CF
gene by immunoassay comprising:
(a) an antibody which specifically binds to a gene product of a mutant CF gene having a mutation at a protein position selected from the group consisting of protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092;
(b) reagent means for detecting the binding of the antibody to the gene product; and (c) the antibody and reagent means each being present in amounts effective to perform the immunoassay.

38. A kit for assaying for the presence of a mutant CF
gene by immunoassay comprising:
(a) an antibody which specifically binds to a gene product of a mutant CF gene having a mutation at a DNA
sequence position selected from the group consisting of DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and (b) reagent means for detecting the binding of the antibody to the gene product; and (c) the antibody and reagent means each being present in amounts effective to perform the immunoassay.

39. The kit of claim 37 or 38 wherein said reagent means for detecting binding is selected from the group consisting of fluorescence detection, radioactive decay detection, enzyme activity detection or colorimetric detection.

40. A kit for assaying for the presence of a CF gene by hybridization comprising:
(a) an oligonucleotide probe which specifically binds to a mutant CF gene; having a mutation at said protein positions selected from the group consisting of protein positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077 and 1092;
(b) reagent means for detecting the hybridization of the oligonucleotide probe to a mutant CF gene having a mutation at said protein positions; and (c) the probe and reagent means each being present in amounts effective to perform the hybridization assay.

41. A kit for assaying for the presence of a CF gene by hybridization comprising:
(a) an oligonucleotide probe which specifically binds to a mutant CF gene having a mutation at said DNA
sequence positions selected from the group consisting of DNA sequence positions 129, 556, 621+1, 711+1, 1717-1 and 3659;
(b) reagent means for detecting the hybridization of the oligonucleotide probe to a mutant CF gene having a mutation at said DNA sequence positions; and (c) the probe and reagent means each being present in amounts effective to perform the hybridization assay.

42. A heterologous cell system comprising a recombinant cloning vector of claim 9, wherein expression of said vector DNA sequence induces cystic fibrosis symptoms in said cell system.

43. The system of claim 42 wherein said system comprises mammalian cells.

44. The system of claim 43 wherein said mammalian cells are from a rodent.

45. The system of claim 44 wherein said rodent is a mouse.

46. The use of oligonucleotide primers in a polymerase chain reaction (PCR) to amplify a selected exon having a cDNA sequence of Figure 1, the use of oligonucleotide primers from intron portions near the 5' and 3' boundaries of the selected exon of Figure 18.

47. The use of oligonucleotide primers in a polymerase chain reaction of claim 46, the use of oligonucleotide primers B 115-B and 10D and oligonucleotide primers xi-5 and xi-3 of Table 5 where X is the exon number 2, 3, 4, 5, 6a, 6b, 7 through 13, 14a, 14b, 15 and 16, 17a, 17b and 18 through 24.