Variable deletion of exon 9 coding sequences in cystic

The EMBO Journal vol.10 no.6 pp.1355- 1363, 1991

Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium Chin-Shyan Chu1, Bruce C.Trapnell', James J.Murtagh, Jr2, Joel Moss2, Wilfried Dalemans3, Sophie Jallat3, Annick Mercenier3, Andrea Pavirani3, Jean-Pierre Lecocq3, Garry R.Cutting4, William B.Guggino5 and Ronald G.Crystall 1Pulmonary Branch and 2Laboratory of Cellular Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA, 3Transgene SA, Strasbourg, France, 4Department of Pediatrics and Center for Medical Genetics, Johns Hopkins Hospital, Baltimore, MD and 5Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD, USA Reprint requests to Pulmonary Branch, Building 10, Room 6D03, National Institutes of Health, Bethesda, MD 20892, USA Communicated by J.P.Lecocq

The predicted protein domains coded by exons 9-12 and 19-23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide-binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9- mRNA transcripts). In most (9 of 12), the exon 9- transcripts represented c25% of the total CFVlR transcripts. However, in three individuals, the exon 9- transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re-evaluation of the same individuals 2-4 months later showed the same proportions of exon 9- transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon- intron junctions relevant to exon 9 deletion (exon 8-intron 8, intron 8-exon 9, exon 9-intron 9, and intron 9-exon 10) were identical except for the intron 8-exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9- transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype. Key words: cystic fibrosis/epithelium/human/lung/mRNA

splicing Introduction Cystic fibrosis (CF), a common fatal hereditary disorder of Caucasians, is caused by mutations in the CF gene, a 27 exon, 250 kb segment of chromosome 7 (Kerem et al., 1989;

Riordan et al., 1989; Rommens et al., 1989; Cutting et al., 1990a,b; Davies, 1990; White et al., 1990). For convenience, we will refer to the normal CF gene as the 'cystic fibrosis transmembrane conductance regulator' (CFTR) gene. The major manifestations of CF are on the epithelial surface of the bronchial tree and gastrointestinal tract (Boat et al., 1989), sites where the CFTR gene is expressed in epithelial cells. In the normal lung, bronchial epithelial cells contain 1-2 CFTR mRNA transcripts per cell (Trapnell et al., 1991). The promoter of the CFTR gene has the characteristics of that of a 'house-keeping'-type gene, and the gene is transcribed in freshly isolated normal bronchial epithelial cells at a relatively low rate (Yoshimura et al., 1991). The function of CFTR is not completely understood, but there is compelling evidence that, at a minimum, it directly or indirectly modulates epithelial cell secretion of Cl- in response to stimuli that raise intracellular cAMP and/or activate protein kinases (Frizzel et al., 1986; Hwang et al., 1989; Li et al., 1989; Drumm et al., 1990; Rich et al., 1990). Analysis of the coding sequence of the CFTR gene reveals regions of CFTR with the potential to bind ATP [see Figure IA for a diagram of the proposed structure of CFTR (Riordan et al., 1989; Hyde et al., 1990)]. The coding sequences for these putative nucleotide (ATP)-binding folds (NBFs) are found in exons 9-12 and 19-23 (Riordan et al., 1989). Although mutations of the CFTR gene have been observed in individuals with CF in exons other than coding exons for the amino-terminal NBF (NBF1), many cases of CF have alleles with mutations in exons 9-12, suggesting that NBF1 plays a major role in the function of CFTR (Riordan et al., 1989; Kerem et al., 1989, 1990; Davies, 1990). In this regard, 68-76% of alleles of individuals with CF have a deletion of 3 nucleotides resulting in loss of phenylalanine at residue 508 (AF508), a region encoded by sequences in exon 10 (Kerem et al., 1989, 1990; Lemna et al., 1990). Consistent with this concept, respiratory and pancreatic epithelial cell lines derived from individuals homozygous for the AF508 mutation do not demonstrate the normal increase in Cl- secretion in response to stimuli that raise cAMP and/or activate protein kinase C (PKC), but do so when modified by the normal CFTR cDNA (Drumm et al., 1990; Rich et al., 1990). The present study demonstrates that some of the CFTR mRNA transcripts in bronchial epithelial cells of normal individuals lack the sequences for exon 9, a sequence that codes for the first 21 % of NBF1 of the CFTR protein.

Results CFTR mRNA transcripts with deletion of exon 9 Evaluation of CFTR mRNA transcripts from bronchial epithelial cells in the region encompassing exons 7-10 after conversion to cDNA and polymerase chain reaction (PCR) amplification demonstrated two different transcripts 1355

C.-S.Chu et al.

(Figure iB). The size difference between these two amplified fragments (509 bp and 326 bp) was the same as the size of exon 9 (183 bp). Southern analysis with an exon 10-specific probe demonstrated that both fragments contained sequences of exon 10 (Figure IC, lane 1). In contrast, Southern analysis with an exon 9-specific probe demonstrated that the 326 bp PCR fragment did not contain sequence of exon 9 (lane 2) i.e. some CFTR mRNA transcripts from bronchial epithelium included exon 9 sequences while others did not. Sequence analysis of the 509 bp fragment (mRNA transcripts appearing to include exon 9 by Southern analysis of PCR fragments) demonstrated that, as expected, mRNA transcripts corresponding to this amplification product included sequences of the 3' end of exon 7, the whole of exons 8 and 9, and the 5' end of exon 10 (Figure 2A; sequences surrounding exon 7-8 junction not shown). Consistent with the Southern analysis, sequence analysis of the 326 bp fragment (mRNA transcripts appearing not to contain exon 9) demonstrated that CFTR mRNA transcripts represented by this fragment did indeed have a deletion of

exon 9, and that the deletion was complete, with in-frame joining of exon 8 to exon 10 (panel B).

Variable deletion of exon 9 in CFTR mRNA transcripts Evaluation of artificial mixtures of CFTR transcripts containing exon 9 ('exon 9+' transcripts) and CFTR transcripts not containing exon 9 ('exon 9' transcripts) demonstrated that input of different relative proportions of exon 9+ and 9- CFTR transcripts in the PCR reaction resulted in the same relative proportions when analyzed after amplification (Figure 3A). Using a similar technique, evaluation of mRNA from bronchial epithelium of 12 normal individuals demonstrated a wide variation in the relative amount of exon 9 + and exon 9- transcripts present (Figures 3B and 4; Table 1). Most individuals had many more exon 9+ transcripts than exon 9- transcripts, with exon 9transcripts only making up 9-25 % of the total. However, three individuals (Figure 3B, lanes 4, 9 and 10) had . 39% exon 9- transcripts. Strikingly two of these individuals had more exon 9- transcripts than exon 9 + transcripts (lanes 4

Fig. 1. Deletion of exon 9 in CFTR mRNA transcripts in normal human bronchial epithelial cells. A. Diagram of the putative CFTR and the exon structure of the mature 27 exon CFTR mRNA (Rommens et al., 1989; Riordan et al., 1989). The predicted domains of CFTR are shown, including membrane-spanning domains, nucleotide-binding folds (NBF) 1 and 2 and the cytoplasmic R domain, with each domain aligned above the mRNA exon sequences which encode the region. The location of the mutation for the common abnormal allele associated with CF, AF508, is indicated in exon 10 and within the NBF1 region. Deletion of exon 9 (Aexon 9) would delete 21% of the amino terminal end of the NBF1 region. The start (ATG) and stop (TAG) codons are shown in exons 1 and 24 respectively. B. Exons 7-10 of the CFTR gene and the two forms of mRNA derived from this region: 'exon 9+' mRNA which contains exon 9 sequences, and 'exon 9' niRNA in which exon 9 has been deleted. Shown are the primers used to amplify the different forms of mRNA (after conversion to cDNA) using PCR, the size of the expected PCR fragments for each mRNA species, and the location of the 'exon 9' and 'exon 10' probes used to evaluate amplified mRNA transcripts. C. Evaluation of the different forms of CFTR mRNA transcripts from freshly isolated, normal human bronchial epithelial cells. Following recovery of the cells, total RNA was extracted, converted to cDNA and sequences including CFTR exons 7-10 amplified using PCR as outlined above. The amplified CFTR transcripts were then separated by agarose gel electrophoresis and evaluated by Southern blotting using exon 9 and exon 10 32P-labeled probes. Lane 1: exon 10 probe detects two species corresponding to exon 9+ and 9- mRNA transcripts. Lane 2: exon 9 probe showing that the smaller transcript does not contain exon 9.

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Exon deletion in CFTR gene mRNA transcripts

and 9), individual 4 having 62% exon 9- transcripts and individual 9 having 66% exon 9- transcripts. To ensure that these observations were reproducible, individuals 1, 4, 6 and 9 were re-evaluated 11 1+ 5 weeks after first evaluation using fresh reagents; all had similar relative amounts of exon 9- transcripts to those found at the first evaluation (Figure 4). There was no correlation between the proportion of exon 9- transcripts and age or sex in the study population (Table I). Sequence analysis of the exon - intron boundaries relevant to deletion of exon 9 of CFTR gene To explore the possible cause of variable deletion of exon 9 in CFTR mRNA transcripts in normal bronchial epithelium, a minimum of 50 bp of the CFTR gene intron sequences relevant to deletion of exon 9 was determined for all 12 individuals in the study population, including the exon 8 -intron 8 junction, the intron 8 -exon 9 junction, the exon 9-intron 9 junction and the intron 9-exon 10 junction (Figure 5; Table II). All 24 alleles in 12 individuals had identical sequences for 50 bp of the intron at the exon 8 -intron 8 junction, the exon 9 -intron 9 junction, and the intron 9-exon 10 junction. However, the region encompassing the intron 8 -exon 9 junction demonstrated interesting allelic intron sequence divergence among the

study population, particularly in the region (probably not critical for the splicing process) between the splice branch site consensus sequence (Krainer and Maniatis, 1988) and the splice acceptor site consensus sequence (Padgett et al., 1986) and for a few individuals, the splice acceptor site consensus sequence itself (Table II). The sequence of the intron 8-exon 9 junction had the same overall structure for all 24 alleles-a splice branch site consensus sequence, a run of 10-12 TG dinucleotide repeats, followed by a splice acceptor site consensus sequence that included a run of T nucleotides ending 6 bp from the start of the exon 9 coding sequence. For all 24 alleles, the putative splice branch site consensus sequence was identical. The length of the TG dinucleotide tract between the splice branch site consensus sequence and the splice acceptor site consensus sequence varied from 10 to 12; most (18 of 24) alleles contained 11 repeats, four alleles had 10 repeats, and two had 12 repeats. The length of the T nucleotide tract in the splice acceptor site sequence varied from 5 to 9 nucleotides; most (21 of 24) alleles contained seven T nucleotides, two alleles had five nucleotides, and one had nine nucleotides. In terms of overall structure (16 of 24 alleles), there were 11 TG dinucleotide repeats in the junction between the branch site sequence and the acceptor site sequence and seven T nucleotides in the acceptor site

4i

a. *.:

. .s:r

Fig. 2. Sequence analysis of exon 9+ and exon 9- CFTR mRNA transcripts of the CFTR gene present in normal human bronchial epithelial cells. A. Sequence of the exon-exon boundary regions flanking exon 9 for exon 9t mRNA. Total RNA was extracted from freshly isolated normal human bronchial epithelial cells, converted to cDNA and sequences including CFTR exons 7-10 were amplified by PCR. After amplification, the PCR products were separated by agarose gel electrophoresis and exon 9+ mRNA-derived transcripts were recovered from the gel, purified and further amplified using the primer combinations shown. The antisense primers (CF84 and CF3) were linked to biotin (indicated by '-O'). The biotinylated strand was then isolated using streptavidin-bound magnetic beads and a magnetic particle concentrator and subjected to dideoxy chaintermination sequence analysis while still coupled to the beads. Shown are the regions surrounding the exon 8-exon 9 junction (left) and the region surrounding the exon 9-exon 10 junction (right). The sequence in this region is identical to that reported for the normal CFTR gene (Riordan et al., 1989). B. Sequence of the exon-exon boundary region for the exon 8-exon 10 junction for exon 9- mRNA. The approach was identical as for panel A except that exon 9- mRNA-derived transcripts were recovered and evaluated using primers in exon 7 and 10 as indicated. The sequence shows that the deletion of exon 9 was complete, with in-frame joining of exon 8 to exon 10.

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sequence. The next most common (three of 24 alleles) contained 10 TG repeats and seven T nucleotides. Other alleles contained 12 TG repeats and seven T nucleotides (two of 24), ten TG repeats and nine T nucleotides (one of 24), or 11 TG repeats and five T nucleotides (two of 24). Strikingly, the two alleles with only five T nucleotides in the acceptor site sequence (one allele each in individuals 4 and 9) correlated with expression of high amounts of alternatively spliced exon 9- CFTR mRNA (Table I). Otherwise, these two alleles had identical intron sequences for the 50 bp of the intron 8-exon 9 junction, including a run of 11 TG repeats in the branch site - acceptor site junction. For convenience, because 21 of the 24 alleles had seven T nucleotides in this region, we will refer to the allele with five T nucleotides in intron 8 -exon 9 of the acceptor sequence as the ATT allele (although theoretically the TT

deletion could be anywhere within the run of seven T nucleotides in a parental allele of 11 TG repeats and seven T nucleotides, or as a T - G conversion in the T nucleotide tract 11 nucleotides from the start of exon 9 coding sequences in a parental allele of 10 TG repeats and seven T nucleotides). Importantly, while comparison to sequences of numerous other eukaryotic and viral mRNA precursors shows that while the TG tract lies within a spacer region of variable length (from 2 to 21 bases) which separates the splice acceptor site and splice branch site consensus sequences, the run of seven T nucleotides lies within a highly conserved (81-91 %) region of the splice acceptor site consensus sequence. Further, the ATT allele has a G 80[z

E

60h-

IC

060

40F-

z E C

20 1-

0

IA4it

oa 0 uJ

0

Initial evaluation

Repeat

evaluation

Fig. 4. Proportion of exon 9- CFTR mRNA transcripts in bronchial epithelial cells from normal individuals. The data are based on the data in Figure 3B. The autoradiograms were scanned by laser densitometry.

Shown is the relative proportion of exon 9 mRNA at the time of initial evaluation of 12 normals (each indicated by a separate data point). Four of these individuals (A, *, V, *) were re-evaluated 11 + 5 weeks later. Note the presence of a large proportion of exon 9- mRNA in epithelium from individuals A, * and 0; data for the initial evaluation are from lanes 4, 9 and 10 respectively of Figure 3B. Re-evaluation of the 4 individuals an average of 11 weeks later showed that the relative proportion of exon 9- mRNA does not change with time in individuals with high or low proportion of exon 9- mRNA transcripts.

Table I. Study population characteristics and proportion of CFTR mRNA transcripts with exon 9 deleted in normal human bronchial cells

Fig. 3. Quantification of the relative amount of alternative deletion of exon 9 of CFTR mRNA transcripts in normal human bronchial epithelial cells. A. Quantification of an artificial mixture of various proportions of exon 9+ and 9- CFTR transcripts using PCR. CFTR exon 9+ mRNA- and exon 9- mRNA-derived transcripts were amplified as outlined for Figure IB, separated by gel electrophoresis, recovered and quantified by optical density. The two species were mixed in various proportions, amplified and the PCR products evaluated by Southern blotting using the exon 10 probe as in Figure IC. The relative proportions of exon 9+ and exon 9transcripts were determined by laser densitometry of the autoradiograms. B. Evaluation of the relative amount of variable deletion of exon 9 in bronchial epithelial cells of 12 normal individuals. Bronchial epithelial cell total RNA was extracted, converted to cDNA, amplified and subjected to Southern analysis using a 32P-labeled exon 10 probe as described in the legend to Figure 1. Each lane represents a different individual. Note that most show a low proportion of exon 9- mRNA, but three individuals (lanes 4, 9 and 10) show a high proportion of exon 9- mRNA.

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Study individual

Age (years)

1 2 3 4 5 6 7 8 9 10 11 12

52 20 29 25 42 42 44 21 25 27 19 19

aData based on Figures 3 and 4.

Sex

Exon 9- mRNA (% of total CFTR transcripts)a

F

9 15 9 62 13 22 25 18 66 39 16 12

M M M M M F F F M M F

Exon deletion in CFTR gene mRNA transcripts A

..EmgI .3 W-

-S

1--,

.5*

4* ;-

Fig. 5. Cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences surrounding the exon-intron junctions of exons 8, 9 and 10. A. Diagram of sequencing strategy. Leukocyte genomic DNA was purified and regions including and surrounding exons 8, 9 and 10 were amplified using pairs of nested primers as indicated. In some amplifications, one primer of the primer pair was coupled to biotin (indicated by '-0' next to primer). After amplification, the biotinylated strand was recovered using streptavidin-bound magnetic beads and a magnetic particle concentrator and subjected to dideoxy chain-termination sequence analysis. The same primers used for PCR amplification as well as internal primers were used to initiate sequence reactions. As shown in panels B -F and summarized in Table HI, the sequences surrounding boundaries examined were all identical except the region of intron 8 - exon 9 junction. There was a 'TT' deletion (ATT) within the T nucleotide tract five bases from the exon - intron boundary in one allele of the two individuals expressing the highest proportion of exon 9- mRNA transcripts. B. Sequence of the exon 8-intron 8 junction observed in all 24 alleles. C. Sequence of the intron 8 - exon 9 junction which was observed in 21 of 24 alleles. D. Sequence of the intron 8-exon 9 junction which was observed in two alleles from two individuals, both of whom had a high proportion of exon 9- mRNA transcripts. Note the deletion of 'TT' which shifts the sequence of one allele (the ATT allele) resulting in overlapping bases and a 3' shift of the repeating TG sequence. E. Sequence of the exon 9-intron 9 junction observed in all 24 alleles. F. Sequences of the intron 9 -exon 10 junction observed in all 24 alleles.

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C.-S.Chu et al. Table HI. Nucleotide sequences surrounding the exon-intron splice junction sites for exons 8, 9 and 10 of the CFTR genea Splice donor site

Study

consensus

sequenceb

Splice branch site

consensus

sequencec

Splice acceptor site

consensus

sequenceb

individual

A62G077! Exon 8 ..A G

G0tsoT10 A6 A74G084T50 Intron 8 G T C

Y1N

A G A

...

-...

I

Y,se

T88 R81 A1oo Y94 N(2-21)

Y78 Y81 Y8 "'89 Y8 Y82 Y81 Y86 Y91 Y87 N Y97 AIse Gis IG5

T G A

T

-

...

T

TT

T

G (TG)9

....~~~~~~~(TG)9

3

..-j..

..(TG)9

4

..-j..

..(TG)9

9

..-I..

..(TG)9

I-... ....

T

A

Intron 8 AC A G

-Exon 9

IG0...

..(TG)8.--I-.---..

....

11

G T T T T T T

-

-

T.-I-.--..

Z I--..--.

.(T G)8....~~~~~~~(TG)9 .

Study individual 1 -12

-Intron 9 Exon 9 ... A 0 G T A 0 T T ... -...

Intron 9

-x40 0 T T T T A T T T CC A ..x4.--

...

A

TT

T

0

A

T

--

G

-Exon I0 IA ... I -...

aData derived from genomic DNA sequences of the 24 alleles of all 12 study individuals. For each study individual (1-12) both alleles are shown. The upper part of the table shows genomic DNA sequences of each allele for the exon 8- intron 8 junction, the putative intron 8 splice branch site, and the intron 8 -exon 9 junction. The lower part of the table shows the corresponding data for the exon 9-intron 9 junction, the putative intron 9 splice branch site, and the intron 9 - exon 10 junction. bConsensus splice donor and splice acceptor sequences determined among - 400 introns of eukaryotic and viral genes encoding proteins (for review see Padgett et al., 1986). The subscripts for the consensus sequence nucleotides denote the frequency of occurrence of the nucleotide shown at that position; Y represents either T or C and R represents either A or G. The sequence for the most common CFTR gene allele is given. Other alleles are listed below; identity with the common allele is indicated by a dash. The deletion of a single nucleotide is indicated by A (T in both examples here). cConsensus splice branch site sequences derived from 16 vertebrate and viral genes after mapping the position of the splice branch site for pre-mRNAs processed in HeLa cell extract or in vivo (Krainer and Maniatis, 1988). The position of the mRNA branch point of the lariat structure formed by the splicing reaction is indicated by * (for review see Krainer and Maniatis, 1988). dX represents the genomic DNA sequence separating the putative intron 9 splice branch site from the intron 9 - exon 9 splice acceptor site sequence. The sequence represented by X (5'-AATGACCTAATAATGATG-3') was identical in all alleles of all individuals.

nucleotide at a position usually (89 %) occupied by a pyrimidine (T in this case) and thus, it might be expected to affect the splicing of exon 9. Discussion The predicted structure of NBFI of CFTR, encoded by sequences within exons 9-12, is a 154 residue, mostly hydrophilic domain (Riordan et al., 1989). A model for the three-dimensional structure of NBFI1 has been developed (Hyde et al., 1990) by aligning the members of the superfamily, predicting the secondary structures for each, combining these predictions into the consensus secondary structure, and developing a three-dimensional structure based on the similarity to the secondary structure of adenylate kinase. NBF1 contains two consensus sequence motifs

capable of binding ATP including a sequence homologous to Walker motif A [amino acid single letter code consensus sequence G-X-X-X-X-G-K-T, where X represents any am-ino acid (Walker et al., 1982); G-S-T-G-A-G-K-T in CFTR (Riordan et al., 1989)] which spans the seven 3' amino acids encoded by exon 9 and the first residue of exon 10, a region homologous to the 'P-loop' consensus motif in ATP- and GTP-binding proteins (Saraste et al., 1990). Another is homologous to Walker motif B [consensus sequence N-NN-N-D, where Ns represent hydrophobic residues thought to line the nucleotide binding pocket (Walker et al., 1982); L-Y-L-L-D in CFTR (Riordan et al., 1989)] located within exon 12. It is likely that deletion of exon 9 would significantly interfere with the binding of nucleotides to 1360

CFTR, thus altering its potential energy-utilizing capacity and possibly the overall function of CFTR. In the context of these considerations, it is difficult to see how CFTR could function without a complete NBFI, and thus without the sequences encoded by exon 9 encompassing 21 % of NBF1L If that is the case, then our observation that there are normal individuals with >60% exon 9- CFTR mRNA transcripts in bronchial epithelium, suggests that only 35-40% of exon 9' CFTR mRNA transcripts are necessary to maintain a 'normal' stat'e in regard to CFTR function in the airway epithelium. This observation helps to define a 'threshold' of normal CFTR mRNA levels necessary to achieve a clinically normal phenotype with respect to CF. This is consistent with our previous demonstration that respiratory epithelial cells from heterozygous carriers of the common abnormal AF508 CFTR gene mutation express equal amounts of normal and abnormal CFTR mRNA transcripts (Crystal et al., 1990). Assuming that mRNA transcripts containing the common AF508 mutation result in CFTR molecules that cannot provide normal CFTR functions, since heterozygotes all have a normal clinical phenotype, only 50 % of the normal CFTR transcripts are needed to provide sufficient amounts of normally functioning CFTR.

Underlying basis of the deletion of exon 9 in CFTR mRNA transcripts The observation that exon 9 ic, precisely deleted from a proportion of the CFTR mRNAtranscripts in the bronchial

Exon deletion in CFTR gene mRNA transcripts

epithelium of normal individuals, yet is present in the genome of these same individuals, makes it likely that exon 9 is deleted from CFTR mRNA transcripts by alternative splicing. The exon 9- transcripts could not be secondary to complete deletion of exon 9 from one allele, since two different copies of intron 8- exon 9 were detected within genomic DNA of individuals whose epithelium expressed both exon 9+ and exon 9- CFTR mRNA transcripts. Moreover, RNA editing, a post-transcriptional process in which an mRNA transcript is altered by addition or deletion of bases, is not a likely explanation as this process is usually limited to alteration of small numbers of bases [such as the observed post-transcriptional change of C to U in apolipoprotein B mRNA (Powell et al., 1987)] or more extensive, albeit apparently unordered, addition of bases to the untranslated region of some mRNA transcripts [e.g., random, post-transcriptional addition of uridine residues to the 3' end of Trypanosoma brucei COIH mRNA (Feagin et al., 1988)]. Assuming alternative splicing to be the mechanism for the deletion of exon 9 in CFTR transcripts, given the wide variation of exon 9 splicing among normal individuals, yet the consistency within each individual over time, there are likely specific mechanisms that control the relative proportion of exon 9+ and exon 9- CFTR transcripts. By analogy to other genes, a likely explanation would be alteration of the specific sequences involved in the splicing process, such as the splice donor consensus sequence, the splice acceptor consensus sequence, or the intron splice branch site consensus sequence (Padgett et al., 1986; Krainer and Maniatis, 1988). The current models for the splicing process suggest that the 5' end of introns in mammalian pre-mRNA binds to the small nuclear RNA (snRNA) Ul in the formation of the 'spliceosomes' which regulate splicing (Krainer and Maniatis, 1988; Guthrie and Patterson, 1988). Other snRNA molecules are involved in the binding of intron sequences at the 3' end (e.g. U5 with the splice acceptor) and at internal positions within the intron (e.g. U2 with the branch site). Mutations in these regions of the intron can reduce the efficiency of utilization of splice sites resulting in alternative splicing (i.e. the deletion of an exon from the mature mRNA). Interestingly, a T - G conversion in the pyrimidine tract of the splice acceptor site consensus sequence at the 3' end of the large intervening sequence of ,3 globin gene, results in (3 thalassemia secondary to reduce normal ( globin InRNA due to decreased efficiency of splicing (Beldjord et al., 1988). In this context, it is interesting that no sequence changes were found in the splice donor sequences for exons 8 or 9 in either allele of any individuals, whether they had a low or high proportion of exon 9- transcripts. Similarly, genomic sequences were identical in all individuals in the region of the exon 10 splice acceptor site sequence. In contrast, although the intron sequence 5' to exon 9 in the region of the splicing branch site sequence showed no mutations in all alleles evaluated, evaluation of the genomic sequences in the region of the exon 9 splice acceptor site showed a TT dinucleotide deletion of one CFTR allele in each of the two individuals who had the highest proportion of exon 9- CFTR mRNA transcripts among the study population. The deletion occurred within a highly conserved [81-91 % based on a review of 400 introns of eukaryotic and viral genes (Padgett et al., 1986)] region of the exon 9 splice acceptor site six bases from the start of exon 9 coding

sequences. In the context that this region is thought to be involved in snRNA binding and spliceosome formation (Krainer and Maniatis, 1988), this observation provides a possible explanation for the higher proportion of exon 9CFTR mRNA transcripts in these individuals. In support of the TT deletion in relation to decreased utilization of the CFTR gene exon 9 splice acceptor site, studies in yeast have demonstrated that the insertion of the thymidine-rich tract preceding a PyAG in an actin gene greatly enhances its ability to be a splice acceptor site (Patterson and Guthrie, 1991). If this is the mechanism, it predicts that most, if not all, bronchial epithelial CFTR transcripts of an individual homozygous for the TT deletion in the intron just 5' to exon 9 would be exon 9-. Independent of the importance of this TT deletion in increasing the probability of having exon 9- transcripts, it probably is not the only mechanism involved. In this regard, of the individuals in the study group, individuals without this TT deletion in one allele had 9-39% exon 9- mRNA transcripts in bronchial epithelium. It is possible, for example, that there are variations in sequences at some other site, probably at other locations not sequences within the 2.2 kb intron 8. Finally, it is important to note that the frequency of the ATT CFTR allele (8 % in our sample) may actually be different in the general population; a larger sampling will be required to assess its frequency accurately.

Materials and methods Study populations Normal, non-smoking individuals [n = 12, 7 males, 5 females, age 30 + 3 years (all data are presented as mean standard error of the mean)] had no history of pulmonary disease or family history of cystic fibrosis. All had normal chest X-ray, pulmonary function tests and normal sweat chloride tests. DNA sequence analysis showed that no individual contains a SF508 CFTR gene allele. Isolation of bronchial epithelial cells Normal bronchial epithelium was obtained from mainstem or lobar bronchi by fiber-optic bronchoscopy, using a 1 mm cytology brush. The sample was immediately suspended in RPMI 1640 (Whittaker Bioproducts). The cells were collected by centrifugation and lysed in 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M j3mercaptoethanol. To ensure the reproducibility of the observations, the bronchial epithelial cells from four individuals were obtained twice, at an interval of 11 + 5 weeks from the initial evaluation.

Evaluation of exon 9 in CFTR mRNA transcripts Total RNA was extracted from the bronchial epithelial cell lysates by the acid guanidinium thiocyanate-phenol -chloroform method (Chomczynski and Sacchi, 1987). The cDNA was synthesized, using Moloney murine leukemia virus RNase H- reverse transcriptase (MMLV H- RT; Gibco/BRL) and random hexanucleotide primer (Pharmacia-LKB), in 40 yd of reverse transcriptase (RT) buffer (50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 0.5 mM each dATP, dGTP, dCTP and dTTP). All oligonucleotides were synthesized using an automated DNA synthesizer (Applied Biosystems) and purified using Sephadex G-25M chromatography (PD-10; Pharmacia). Some oligonucleotides were biotinylated using biotin phosphoramidite (DuPont) according to manufacturer's protocols. All polymerase chain reaction (PCR) amplifications were performed using the DNA Thermal Cycler (Perkin-Elmer Cetus). Trnscripts of the CFTR gene were amplified by PCR using recombinant Taq DNA polymerase [AmpliTaq; Perkin-Elmer Cetus (Saiki et al., 1988)]. The oligonucleotide primers included 5' primer CF37 (5'-CAGAACTGAAACTGACTCGGAAGG-3', located in exon 7) and 3' primer CF31 (5'-TCTTTCTCTGCAAACTTGGAGATG-3', located in exon 1 1) and amplification was performed for 10 cycles (94°C, 20 s; 64°C, 20 s; 72°C, 60 s). Further 'nested' amplification was performed on a fixed aliquot (5%) of the first PCR reaction

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C.-S.Chu et al. under similar conditions, except that CF85 (5'-ACAAACATGGTATGACTCTCTTGG-3', located in exon 7) and CF3 (5'-CTTCTAGTTGGCATGCTTTGATGACGCTTC-3', located in exon 10) were used for 25-30 cycles. Ten percent of the total PCR products were size fractionated on 1.5% agarose gel in I x TBE buffer (89 mM Tris, pH 8.3, 89 mM boric acid, 2 mM ethylenediaminetetraacetate). Duplicate gels were blotted onto Nytran membranes (Schleicher & Schuell) by the method of Southern (Southern, 1975). DNA was fixed to the filter by UV irradiation (UV Stratalinker; Stratagene). One filter was hybridized with a 125 bp 32Plabeled 'nested' CFTR cDNA probe located in exon 10 ('exon 10' probe), washed and evaluated by autoradiography. The other filter was hybridized with a 177 bp 32P-labeled 'nested' CFTR cDNA probe located in exon 9 ('exon 9' probe; see Figure lB for location of all probes). Nucleotide sequence of CFTR transcripts from normal human bronchial epithelial cells After preliminary observations of the PCR products suggested that two different CFTR transcripts were present [509 bp ('exon 9+' transcripts) and 326 bp ('exon 9' transcripts)], bronchial epithelial cells from each individual were obtained by brushing, total RNA was extracted, converted to cDNA, amplified by PCR and size fractionated by agarose gel electrophoresis. The fragments of exon 9+ and exon 9- transcripts were excised from the agarose gel and purified (Geneclean II; Bio 101) according to protocols supplied by the manufacturer. The exon 9- transcripts were further amplified by using primers CF85 and CF3-O (see above; '-O' indicates that the primer was biotinylated) for 30 cycles. Biotinylated singlestranded CFTR transcripts were prepared by using a magnetic particle concentrator and Dynabeads M-280 streptavidin (Dynal) according to manufacturer's protocols. Briefly, streptavidin is a protein with four high affinity binding sites for biotin. Dynabeads M-280 streptavidin are superparamagnetic, polystyrene beads with chemically bound high quality streptavidin. To obtain single stranded CFTR transcripts, Dynabeads M-280 streptavidin were added to above PCR products containing biotinylated transcripts. After the supernatant was removed using the magnetic particle concentrator, 0.2 M NaOH was added to convert double-stranded cDNA to single-stranded cDNA. After removing the supernatant, the single-stranded CFTR transcripts containing CF3-O were sequenced by the dideoxy chaintermination method (Sequenase; USB) using CF85 (Hultman et al., 1989). The exon 9+ transcripts were sequenced using the same technique as above except that two separate sets of PCR and sequencing primers were used: (i) PCR with CF85 (see above) and CF84-O (5'-CTGCTCCAGTGGATCCAGCAACCG-3', located in exon 9) and sequencing with CF85; and (ii) PCR with CF39 (5'-CTTCAGTAATTTCTCACTTCTTGG-3', located in exon 9) and CF3-O (see above) and sequencing with CF39.

Quantification of exon

9+ and exon 9-

CFTR mRNA

transcripts epithelial cell total RNA was extracted, converted to cDNA, amplified by PCR and subjected to Southern analysis using a 32P-labeled exon 10 probe described as above. The density of cDNA fragments on the autoradiogram was measured by LKB 2202 UltroScan laser densitometer (Pharmacia-LKB), and the relative amount of exon 9+ and 9- transcripts (as a percentage of total CFTR transcripts) was calculated using the total densitometric units of both transcripts as 100%. To establish that the PCR amplification accurately reflected the relative amounts of exon 9+ and exon 9- transcripts initially present, variable amounts of exon 9+ and 9- transcripts were mixed (total of 103 transcripts in each PCR reaction) and amplified with primers CF85 and CF3 (see above) for 27 cycles. To accomplish this, the concentration (jsg/ml) of purified exon 9+ and 9- transcripts was measured using a DU-70 Spectrophotometer (Beckman) and the molecular weight calculated using the number of base pairs of each cDNA segment (exon 9+ transcripts: 509 bp; exon 9- transcripts: 326 bp). The PCR amplification products of these mixtures were evaluated by Southern blotting and autoradiography as Bronchial

described above.

Nucleotide sequence of the CFTR gene relevant to exon 9 deletion Human genomic DNA from each individual was prepared from peripheral leukocytes. The regions flanking the 3' end of exon 8, 5' and 3' ends of exon 9 and 5' end of exon 10 were amplified by PCR as described above using primers specific for each region (Figure 5A). For each region, a minimum of 50 bp of intron sequences was determined for both alleles of all 12 individuals. The single-stranded genomic DNA was prepared and sequenced by the dideoxy chain-termination method as above. For the convenience of discussion, we have numbered the introns 1-26 beginning with the intron 3' to exon 1 (Rommens et al., 1989; Riordan et al., 1989).

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The region surrounding 3' end of exon 8 was amplified with CFI13 (5'-AATCCTAGTGCTTGGCAAATTAAC-3', located in intron 7, oligonucleotide primers located in CFTR gene introns were based on sequence data provided by Zielenski,J., Rozmahel,R., Bozon,D., Kerem,B.-S., Grzelczak,Z., Riordan,J.R., Rommens,J. and Tsui,L.-C., in preparation) and CFI12 (5'-TCGCCATTAGGATGAAATCC-3', located in intron 8). Further partially 'nested' amplification was performed on the products of the first PCR reaction with CF97 (5'-CTTACAAAAGCAAGAATATAAGAC-3', located in exon 8) and CFI12-0. Both amplifications were performed under similar conditions (94°C, 20 s; 55°C, 20 s; 72°C, 60 s) except that 25 cycles were performed for first amplification and 30 cycles for second amplification. Biotinylated single-stranded genomic DNA containing CFI12-0 was sequenced with CF97. The sequence of the region surrounding the intron 8-exon 9 junction for one strand of DNA was determined by: (i) first PCR with CFI1 (5'-TTGATAATGGGCAAATATCin located intron and CFI2 TTAG-3', 8) (5'-AAGATACAGTGTTGAATGTGGTG-3', located in intron 9); (ii) second PCR with CFI3-0 (5'-TTAGATCATGTCCTCTAGAAACCG-3', located in intron 8) and CFI4 (5'-GTGGTGCAAAATATAAAGATGTGG-3', located in intron 9); and (iii) sequencing with CF38 (5'-GAAATTAATATCTTTCAGGACAGG-3', located in exon 9). The sequence of the opposite strand in the same region was determined by: (i) first PCR with CFI1 and CFI2; (ii) second PCR with CFI3 and CFI4-0; (iii) sequencing with CFI25 (5'-TGTATACAGTGTAATGGATCATGG-3', located in intron 8). The sequence of the region surrounding the exon 9-intron 9 junction was determined by: (i) first PCR with CFI1 and CFI2;

(ii) second PCR with CFI3 and CFI4-0; and (iii) sequencing with CF39. The sequence of the region surrounding the intron 9 -exon 10 junction was determined by: (i) first PCR with CFIR1 (5'-TAAGAATATACACTTCTGCTTAGG-3', located in intron 9) and CF3; (ii) second PCR with CFIl 1 and CF74-0 (5'-GATATTTTCTTTAATGGTGCCAGG-3', located in exon 10); and (iii) sequencing with CFI 1.

Acknowledgements We would like to thank Milica S.Cherniack, National Institute of Diabetes, Digestive and Kidney Diseases for help with the clinical evaluation of the study group and Theresa Raymer for editorial assistance. This work was supported in part, by the Cystic Fibrosis Foundation (USA), and l'Association Fran~aise de Lutte contre la Mucoviscidose (France).

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