Hum Genet (1992) 88 : 552-556

9 Springer-Verlag1992

Screening for cystic fibrosis gene mutations by multiplex DNA amplification Luigi Picci, Franca Anglani, Maurizio Scarpa, and Franco Zacchello Department of Pediatrics, University of Padua, Via Giustiniani, 3, 1-35128 Padua, Italy Received February 8, 1991 / Revised June 20, 1991

Summary. We have developed a simple rapid D N A screening test that allows us simultaneously to analyze seven CF mutations (deltaF508, R347P, $549N, G551D, R553X, R334W, 444delA) that together account for about 60% of all CF mutations in the Italian population. It consists of three steps: multiplex polymerase chain reaction (PCR) amplification of exons 4, 7, 10 and 11; restriction endonuclease digestion of the P C R products; and vertical polyacrylamide gel electrophoresis analysis. We have used our multiplex assay for analyzing 15 CF chromosomes (non delta F508) and have found 3 cases of the R553X mutation; the latter have been confirmed by amplification and digestion of exon 11.

Introduction The cloning of the cystic fibrosis (CF) gene and the identification of the most c o m m o n mutation, deltaF508 (Kerem et al. 1989), provide a basis for understanding the pathophysiology of the disease, and for the development of a more efficient therapy and carrier identification. Many additional mutations producing CF have however been identified to date. The identification of multiple, individually rare mutations rather than a small number of c o m m o n mutations, makes carrier testing more difficult. There is a consensus that under the current circumstances, population-based screening should not be r e c o m m e n d e d for individuals and couples with a negative family history (Workshop on Population Screening for Cystic Fibrosis Gene 1990). Whatever the present limitations of mutation screening on a large scale, it is important to develop procedures that facilitate the techniques and lower the cost of D N A testing. Multiplex D N A amplification fulfills these two criteria, because widely separated sequences can be amplified in only one reaction. Multiplex D N A amplification has been used to analyze deletions at various loci (Chamberlain et al. 1988; Ballabio et al. 1990; Gibbs et al. 1990). We have developed a multiplex assay to analyze point mutations in the CF gene. It provides a rapid simple

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method of simultaneously analyzing the mutation delta F508 (Kerem et al. 1989), and 6 point mutations that alter restriction enzyme sites: R347P (Dean et al. 1990), $549N, G551D, R553X (Cutting et al. 1990), R334W (Gasparini et al. 1991) and 444delA (White et al. 1991).

Materials and methods D N A templates for polymerase chain reaction amplification DNA of normal, CF-heterozygous and CF-affected individuals was obtained from 10 ml blood by standard methods (Buffone and Darlington 1985) or from 50 gl blood by a slight modification of the procedure of Kawasaki (1990).

Multiplex amplification reaction The coamplification of the exons 4, 7, 10 and 11 of the CF gene was achived by using eight pairs of oligonucleotides in a single polymerase chain reaction (PCR). Two of them were derived from intron flanking sequences (11i-3 and 11i-5) and six from exon sequences. The primer sequences, the sources, and the size of the amplified regions are listed in Table 1. Coamplification was usually carried out with 200 ng genomic DNA as template. Templates were mixed with primers in a total volume of 50gl, containing 6.7mM MgCI2, 16mM (NH4)2804, 10mM 2-mercaptoethanol (BME), 67mM TRIS-HC1 pH 8.8, 8.5gg bovine serum albumin (BSA), 100gM each deoxyribonucleotide triphosphate, and 1.25 U Taq DNA polymerase (Amplitaq; Perkin Elmer Cetus). The final primer concentrations were: 2 pmol for CF-32 and CF-33, 8pmol for CF-34 and CF-35, 32pmol for Mathew's primers (Mathew et al. 1989) and 16pmol for 11i-3 and 11i-5. The reactions were overlaid with mineral oil and heated for 5min at 94~ followed by 30 cycles of DNA extension (72~ 90s), annealing (54~ 48s), and denaturation (93~ 48s). All PCRs were carried out in a Perkin Elmer Cetus DNA Thermocycler.

Restriction endonuclease digestion The list of the mutations that alter recognition sites is reported in Table 2. Three different enzymatic digestions were set up: one with NcoI/DdeI for the R347P and $549N mutations; one with HindII/MspI for the G551D, R334W and R553X mutations; and one with Sau3A for the G551D and 444delA mutations. A 10-gl sample from the PCR reaction was incubated overnight at 37~ with 2 U enzyme in the appropriate digestion buffer.

553 Table 1. Oligonucleotide primers for the multiplex amplification Exon 4 7 10 11

Sequence primer F R F R F R F R

5'-AGT 5'-GCT 5'-CAG 5'-TGC 5'-GGC 5'-CTA 5'-CAA 5'-GCA

CAC Aq-T AAC TCC ACC TAT CTG CAG

CAA AGC AGT ACA GC-3' CTC ATC TGC ATT CC-3' TGA AAC TGA CTC GG-3' A A G A G A G T C ATA CC-3' ATT AAA GAA AAT ATC-3' TCA TCA TAG GAAAC-3' TGG TTA A A G C A A TAG TGT-3' ATT CTG AGT AAC CAT AAT-3'

Table 2. Mutations modifying restriction enzyme sites in CF exons

Exon

11

Primer designations

Amplified

Reference

CF-32 CF-33 CF-34 CF-35 -

189 bp

Dean et al. (1990)

230 bp

Dean et al. (1990)

50 bp

Mathew et al. (1989)

11i-5 11i-3

425 bp

Cutting et al. (1990)

Mutation

Nucleotide change

Amino acid change

Restriction-enzyme-site change

444delA

1-bp deletion

Frameshift

Creation of a Sau3A site

R334W R347P

C-1132-*T G-1172---~ C

Arg-334---~Trp Arg-347-->Pro

Loss of an MspI site Creation of an NcoI site

$549N G551D

G-1778--~A G-1784---~A

Ser-549---~Asn Gly-551---~Asp

R553X

C-1789---~T

Arg-553-*Stop

Loss of a DdeI site Loss of a HindII site Creation of a Sau3A site Loss of a HindII site

The restriction map of the amplified regions allowed us to calculate the number and the size of restriction fragments generated by each digestion of the normal and mutated exons (Table 3).

Vertical polyacrylamide gel electrophoresis The digestion products were analyzed by vertical polyacrylamide gel electrophoresis (PAGE). An 8-gl sample of the digestion reaction was applied to a 12% polyacrylamide gel (16 x 16 x 0.075 cm) containing 90 mM TRIS-borate and 10% glycerol. Electrophoresis was performed at 45 mA for 3 h, and the bands were visualized by ethidium bromide staining.

Silver staining To enhance the visualization of the electrophoretic bands and to improve the resolution of PAGE analysis, the gels were stained with silver using a rapid method modified from the procedure of Ohsawa and Ebata (1983). After ethidium bromide staining, gels were soaked twice in water and then placed in 250 ml freshly prepared ammoniacal silver solution (NH3 0.1%, AgNO3 0.1%, NaOH 0.1%). After 20 min, the gels were rinsed in water twice for 30 s and transferred to freshly prepared developer solution containing 0.005% citric acid and 0.02% formaldehyde. The stained DNA became visible after 5 min. Gels were gently agitated in developer solution until the image had reached the desired intensity. Development was stopped by discarding the developer and adding water.

Results and discussion

Multiplex amplification T h e d e v e l o p m e n t of conditions for the coamplification of the 4 C F exons required the sequential addition of individual p r i m e r pairs into a single reaction. Testing

followed to d e t e r m i n e w h e t h e r conditions n e e d e d to be modified to enable a p p r o x i m a t e l y equal amplification of the different fragments. Initially, we chose the cycling p a r a m e t e r s that e n a b l e d us to amplify the small s e g m e n t o f exon 10 (50-bp long), containing the deltaF508 m u t a tion. T h e s e conditions a p p e a r e d to be suitable for the amplification of all the o t h e r exons. It was necessary to adjust the c o n c e n t r a t i o n of each p r i m e r used in the reaction to prevent the f o r m a t i o n o f u n w a n t e d bands. This was achieved by using an unusual p r o p o r t i o n o f p r i m e r pairs. T h e p a t t e r n o f the coamplification of the exons 4, 7, 10, and 11 is illustrated in Fig. 1A. Despite the high c o n c e n t r a t i o n of the D N A t e m p l a t e and the low c o n c e n t r a t i o n s of the primers, b a n d s produced by primer-dimers a p p e a r e d as 50 to 40-bp fragments. F o r m o s t applications, 200 ng g e n o m i c D N A was used as a multiplex template, a l t h o u g h we successfully screened a series of crude cell lysates that were o b t a i n e d f r o m 50 gl blood.

Restriction endonuclease digestion and P A G E analysis T h e preliminary restriction m a p analysis of the amplified C F e x o n regions allowed us to establish that, in each set of digestions, the restriction fragments did not overlap. Thus, each m u t a t i o n could be recognized by characteristic bands. In the NcoI/DdeI digestion, h o w e v e r , the 50-bp f r a g m e n t of e x o n 7 o v e r l a p p e d the 50-bp amplified region o f the n o r m a l exon 10 (Table 3). Vertical 12% p o l y a c r y l a m i d e gels with 10% glycerol allowed us to separate all the restriction fragments o f a wide range o f length (from 457 to 47-bp) with g o o d resolution, in one electrophoretic run. H o w e v e r , some

554 differences between the e x p e c t e d and o b s e r v e d restriction fragments are seen: the size of the largest fragments f r o m each digestion reaction is different from that expected, and s o m e fragments that differ by only a few

Table 3. Expected restriction fragment sizes (bp) generated by enzymatic digestion of normal and mutated exons

Exon

Normal

Digestion Ncof/DdeI 4 7

i89 165

10 11

50 15 50 238 174 13

Digestion Sau3A

Mutation R347P

$549N

189 104a 61a 50 15 50 238 174 13

189 165

444delA 153a 352 230 50 425

189

189

7 10 11

230 50 425

230 50 243a 182a

4 7 10 11

Fig. 1A-D, PAGE analysis of multiplex PCR products (A) and restriction fragments generated by NcoI/DdeI (B), Sau3A (C) and HindII/MspI (D) digestion of amplified DNA from normal individuals. The observed restriction fragment sizes are reported in Table 4. m pBR322 DNA digested with HaeIII

13

G551D 4

Digestion HindII/MspI

50 15 50 412b

R553X

G551D

R334W

133 56 128 102 50

133 56 128 102 50

133 56 128 102 50

133 56 230b

239 186

425b

425b

239 186

a The creation of restriction enzyme sites by the mutations produces two new fragments of a smaller size b The loss of restriction enzyme sites does not alter the size of the amplified region of exons 11 and 7 Table 4. Observed restriction fragment sizes

(bp) generated by the digestion of amplified normal CF exons. The order of the fragments in each column is shown as it appears in the gel after PAGE analysis (see Fig. 1). The expected size of the fragments are reported in parentheses

Exon

NcoI/DdeI

11

267 (239)

4 11 7

7 10

197(189) 185 (174) 170(165)

56 (50) 50 (50)

bases are not resolved (Table 4). Nevertheless, a normal electrophoretic pattern specific for each digestion is recognizable in o u r experimental p r o c e d u r e of P A G E analysis (Fig. 1B, C, D). T h e deltaF508 mutation, recognizable by the a p p e a r a n c e of a 47-bp fragment, is detectable in the electrophoretic run of the N c o I / D d e I digestion. The primer-dimer b a n d (50-bp long) that overlaps the amplified region of exon 10 is digested by these enzymes. T h e expected 50-bp fragment g e n e r a t e d by the N c o I / D d e I digestion of exon 7 migrates as a 56-bp fragment. T h e P A G E analysis of the N c o I / D d e I D N A digestion of deltaF508 heterozygous and h o m o z y g o u s individuals clearly shows the a b n o r m a l 47-bp b a n d (Fig. 2A). To improve the solution of P A G E analysis, a silverstaining m e t h o d was used. Figure 2 shows the higher sensitivity of silver staining (A) in detecting smaller fragments in c o m p a r i s o n with ethidium b r o m i d e staining (B). T h e rapid silver staining p r o c e d u r e m a y be used as a m e t h o d of gel staining or after ethidium b r o m i d e staining to e n h a n c e some weak signals. T h e multiplex assay was tested for the detection of C F mutations using the D N A of C F h e t e r o z y g o t e s that

Exon

Sau3A

11

457(425)

7 4

245 (230) 197 (189)

10

50 (50)

Exon

HindII/MspI

11

267 (239)

11

195 (189)

7&4 7 4 lO

143 (128 & 133) 105 (102) 59 (56) 50 (50)

555

Fig. 2A, B. Silver staining (A) and ethidium bromide staining (B) of polyacrylamide gel loaded with NcoI/DdeI-digested DNA from normal (lane 1), deltaF508 heterozygous (lane 2) and homozygous (lane 3) individuals; m as in Fig. 1

Fig. 4. PAGE analysis of HindII/MspI digestion of 15 CF nondeltaF508 chromosomes. Lanes 2, 9, 13 three cases of R553X mutations; m as in Fig. 1

Conclusion

Fig. 3. PAGE analysis and silver staining of normal (lanes 1-3, 8-10, 15-17) and mutated exons from CF heterozygotes for the $549N (lanes 4, 11, 18), R553X (lanes 5, 12, 19), R334W (lanes 6, 13, 20) and R347P (lanes 7, 14, 21) mutations. The black arrowheads indicate the expected supernumerary bands; m as in Fig. 1

had previously been identified as having the $549N, R553X, R347P and R334W mutations by amplification of a single exon. A PCR with no added D N A template was carried out together with multiplex amplifications to control for the possible introduction of exogenous contaminating sequences that could produce spurious amplification products (Chamberlain et al. 1988, 1989). A clear electrophoretic pattern for each mutation was detected; the extra bands generated by the digestion of the mutated exons with the appropriate enzyme were those expected (Fig. 3). T o test our mutliplex assay further, we analyzed 15 CF non-deltaF508 chromosomes, and found 3 chromosomes carrying the R553X mutation (Fig. 4). The mutation was confirmed by the amplification and digestion of only exon 11.

The application of D N A amplification to the diagnosis of genetic diseases is becoming increasingly common. Multiplex D N A amplification provides a rapid reliable method for screening rearrangements at a particular locus. To date, multiplex assays have been applied to detect deletions; here, we have demonstrated that multiplex P C R can be employed to detect point mutations. By using one amplification, three digestion reactions and one electrophoretic run, we can simultaneously analyze 7 CF mutations that together account for about 60% of all the CF mutations in the Italian population (Devoto et al. 1990). The specificity of our method has been demonstrated by testing previously characterized D N A samples from CF-obligate heterozygotes, and correlates with that of single exon amplification. The sensitivity of our screening test in detecting mutations can be implemented by increasing the number of digestion reactions or by coupling compatible enzymes, and/or combining it with allelespecific oligonucleotide hybridization. Thus, we could detect other CF mutations that are localized in the hot spot of the gene represented by exon 11 (Kerem et al. 1990). The simplicity and specificity of the multiplex assay makes it an ideal system for routine screening of CF mutations in large population samples. Acknowledgements. The authors wish to thank Dr. M. Devoto and Professor G. Romeo for having kindly supplied control DNA for the $549N, R553X, R347P and R334W mutations and the European Community Concerted Action on Cystic Fibrosis for helpful support.

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