Download A Rapid Screening Method to Detect Nonsense and Frameshift

[CANCER RESEARCH 53, 5581-5584, December 1, 1993]
Advances in Brief
A Rapid Screening Method to Detect Nonsense and Frameshift Mutations:
Identification of Disease-causing A P C Alleles
Liliana Varesco, Joanna Groden, 2 Lisa Spirio, Margaret Robertson, Robert Weiss, Viviana Gismondi, G. B. Ferrara,
and Ray White 3
lstituto Nazionale per la Ricerca sul Cancro, Genoa, Italy [L. V, V G., G. B. F..], and Howard Hughes Medical Institute [J. G., M. R., R. Wh.] and Department of Human
Genetics [L. S., R. W., R. Wh.], Eccles Institute of Human Genetics, The University of Utah, Salt Lake City, Utah 84112
Abstract
Materials and M e t h o d s
A functional screen for nonsense and frameshift mutations has been
devised that allows genes of interest to be scanned in segments. This assay
is based on the cloning of these segments in-frame with a colorimetric
marker gene (lacZ) followed by screening for the level of functional activity from the marker polypeptide (fl-galactosidase). Individuals at risk
for any one of a number of genetic diseases, in particular familial adenomatous polyposis coil (APC), can be quickly screened for chain-terminating mutations introduced by stops and frameshifts. At present, scanning of
the APC gene for mutation requires significant effort because it is a large
gene and most APC mutations are unique. Therefore, this assay offers a
powerful option for the diagnosis of this and other genetic diseases, as well
as great potential for the development of a similar rapid screen to detect
APC mutations in colorectai adenomas and carcinomas.
Plasmid Construct and Preparation. The pSTDTAC-IKS plasmid is a
7.3-kilobase plasmid derived from pBR322 that contains the entire lacZ coding
sequence (14). A 300-base pair insert containing multiple in-frame stops is
flanked by ApaI and HindIII cleavage sites. This insertion is located between
expression control sequences that include a Tac promoter, lac operator and
translational start sequences and the/3-galactosidase coding sequence; in-frame
replacement of this out-of-frame insert is required for high levels of expression
of the lacz gene. The plasmid also carries an ampicillin resistance gene.
Plasmid DNA was used to transform host bacterial strain XL1-Blue (Stratagene), which was grown in Terrific Broth and was isolated by means of a
Qiagen plasmid preparation column. The vector was cut with HindlII and ApaI
restriction endonucleases (Boehringer Mannheim) and was gel purified using
Geneclean.
Amplification of Test DNA and Cloning. Template DNA was obtained
from APC patients, normal individuals, and colorectal carcinoma cell line
SW480 (available from the American Type Culture Collection) as described
previously (2, 8). APC mutations in DNAs had already been identified by other
methods (2, 3, 8). Insert DNA was prepared using PCR with the following pair
of oligonucleotide primers: BWL2-GGCTGCAGGAATI'CGATATCAAGCTITG CTGCCCATACACATI'CAAACAC and BWR4-GGCTGCGGAATTCGATATGGG CCCATGAGTGGGGTCTCCTGAAC. These primers amplify 1370 base pairs of APC sequence from nucleotide 2779 to 4151 in exon
15 of the APC gene and include appropriate restriction sites for directional
cloning into pSTDTAQ-IKS as well as nucleotides within BWL2 to adjust the
frame of the DNA sequence produced. (There are no known ApaI or HindlII
restriction sites in this segment of the APC gene.) DNA samples were amplified by the PCR: 5 min at 95~ once; 1 min at 95~ 1 min at 60~ 1 rain at
72~ 30 cycles. Reaction mixtures contained the following: 200-400 ng of
DNA template; 10 ~M each primer; 75 p~Meach deoxynucleotide triphosphate;
10 mM Tris, pH 8.3; 50 mM KCI; 1.5 mM MgCI2; 0.01% gelatin; 2.5 units of Taq
polymerase. For each DNA sample analyzed, six PCR reactions were performed. Aliquots of each of these six PCR reactions were electrophoresed
through a 2% ME agarose gel to check for amplification. The PCR products
were pooled and split into two samples, which were each phenol/chloroform
extracted and ethanol precipitated. Each pellet was washed once with 70%
ethanol and resuspended in 20 /xl of water. PCR products were digested
sequentially with ApaI and HindlII. Enzymes were heat inactivated at 65~
for 20 rain. Digested products were purified with Centricon 100 columns
(Amicon) and electrophoresed to estimate DNA concentration. Products were
mixed at a molar ratio of 1:2 or 1:3 (vector to insert) in a 20-/xl ligation reaction
using a Stratagene ligation kit.
Transformation and Plating. Cloned DNAs (1 /xl of each of the 20 ~1
ligation reactions, or about 30 ng of vector DNA) were introduced into Sure
Electrocompetent Cells (40 ixl) (Stratagene) by electroporation in Bio-Rad
Gene Pulser cuvets. The transformation efficiency of these cells was approximately 5 • 109, as determined by the use of pUC18 as a control. The
electroporated cells were resuspended in 960/xl of S.O.C. medium (Bethesda
Research Laboratories). Aliquots of 250 txl were then plated on each of two LB
agar plates (180 cm) with 100/xg/ml ampicillin, 15/.Lg/ml tetracycline, and 80
/.zg/ml X-gal. IPTG was not included in the plating as the levels of constitutive
expression were adequate for colorimetric determination and high expression
levels appeared to inhibit colony formation. Plates were incubated overnight at
37~ and stored at 4~ to enhance the color of the colonies. Colony color and
numbers were then scored, 2 days and again 4 days after incubation.
Introduction
T h e accurate detection o f mutation in h u m a n t u m o r suppressor
genes is important for the clinical diagnosis of inherited diseases that
predispose to neoplasia. Linkage analysis can follow the inheritance
of these mutant genes within kindreds with a calculated likelihood for
carrier status, but only a direct analysis of the g e n e will determine the
g e n o t y p e of an at-risk individual in the absence of familial analysis.
Therefore, rapid and reliable m e t h o d s for detecting mutation m u s t be
developed.
A P C 4 represents an excellent candidate disease for d e v e l o p i n g such
approaches. Disruption o f the A P C gene leads to a highly penetrant,
inherited f o r m of colon cancer (1-3). Mutational analyses o f the o p e n
reading frame of APC, undertaken by a n u m b e r o f groups, have s h o w n
that virtually all germline mutations are frameshift or point mutations
that result in premature stop c o d o n s (2-11). T h e majority of these
m u t a t i o n s are unique within a given family, with the exception of two
deletions that together represent about 10% o f k n o w n germline m u tations (4, 7, 8, 10, 11). Because the A P C c o d i n g sequence is greater
than 8.5 kilobases, it is extremely difficult to scan or sequence this
gene rapidly (12, 13). Therefore, a functional test has been designed
that allows direct detection of mutations in individuals. This assay is
based on the level of expression of a fl-galactosidase coding sequence
present at the 3' end of a P C R - a m p l i f i e d and cloned D N A s e g m e n t in
this expression vector system.
Received 9/29/93; accepted 10/20/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Howard Hughes Medical Institute, NIH Grant
5P30-HG00199-03, the Associazionc ltaliana Ricerca sul Cancro, C.N.R. Target Project
"Biotechnology and Bioinstrumentation" Contract N. 93.0110PF70, and C.N.R. Progetto
Finalizzato Ingegneria Genetica Contract N. 93.00050.PF99.
2 Present address: Department of Molecular Genetics, Biochemistry and Microbiology,
The University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524. To whom
requests for reprints should be addressed.
3 Investigator of the Howard Hughes Medical Institute.
4 The abbreviations used are: APC, adenomatous polyposis coli; PCR, polymerase
chain reaction; IPTG, isopropyl-13-D-thiogalactopyranoside;X-gal, 5-bromo-4-chloro-3indolyl-t3-o-galactopyranoside.
5581
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SCREEN FOR APC MUTATIONS
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Fig. 1. Three types of colonies that result from
cloning an A P C segment amplified from the DNA
of a normal individual. Clear arrow, blue colony
(in-frame cloning); short black arrow , white
colony (vector background); long black arrow, light
blue colony (translational restart of in-frame cloning or PCR error that changed the frame).
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Mutation Detection and DNA Sequencing. Colonies to be checked for the
sequence of insert DNA were picked from the plates into 100/xl of water and
boiled for 10 min. An aliquot was then PCR amplified with custom APC
primers (also carrying M13 universal or reverse sequencing primers) flanking
the known mutation or with primers that were allele specific for either of the
two common APC deletions (8). Samples for sequencing were purified with
Centricon 100 columns (Amicon) and finally sequenced with an Applied
Biosystems model 373A DNA sequencer, as described previously (2, 8, 9).
,
,.
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in-frame cloning and maintenance of the reading frame in DNA that
has a normal APC sequence; this results from the production of high
levels of functional /3-galactosidase protein. A white colony is produced by plasmid not carrying a cloned DNA insert, resulting in
almost no production of/3-galactosidase. The in-frame cloning of an
insert that contains a sequence with a frameshift or a premature stop
codon, however, produces a colony of intermediate blue color the
intensity of which varies with the specific mutation. This result may
be due to reinitiation at downstream in-frame ATGs in the cloned APC
segment (positions 2845, 2935, 3631, and 4150) or to factors specific
to the plasmid and bacterial strains used in this assay (see "Discussion").
The intermediate blue colonies derive from two types of inserts:
mutations present in the original template DNA and errors generated
by PCR amplification of the insert. This conclusion was supported by
sequencing of dark and intermediate blue colonies derived from the
testing of APC patient 3400. Each of three intermediate blue colonies
sequenced contained the stop mutation found in this patient and each
of three dark blue colonies contained normal APC sequence (data not
Results
A functional assay has been designed to detect mutations that disrupt translation of the A P C reading frame. The assay is composed of
four steps: step 1, PCR amplification of APC sequence from genomic
DNA or cDNA; step 2, cloning of these sequences into a plasmid,
upstream and in-frame, with a/3-galactosidase coding sequence; step
3, transformation of bacteria; and step 4, plating onto X-gal-containing medium and differential counting of blue and white colonies to
indicate the presence or absence of a mutation in the insert.
In practice, three types of bacterial colonies are seen with this
protocol, as shown in Fig. 1. An intense blue colony is produced from
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Fig. 2. Result of three clonings and platings; each DNA sample was prepared from an individual or cell line with a different A P C genotype. Colors and distribution of colonies
containing amplified DNA from (A) an individual with a normal genotype at the A P C locus; (B) premature stop codon in A P C at position 4015; (C) a colorectal carcinoma cell line
(SW480) that is homo- or hemizygous for an A P C stop mutation at position 4017. All three samples are represented numerically in Table 1.
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SCREEN FOR APC MUTATIONS
Table 1 Colony colors and numbers obtained by cloning APC segments from different
templates into the reporter plasmid
Scoring for most samples was done at 2 and 4 days; both numbers are included below
with the 2-day count to the left of the shillingand the 4-day number shownon the right.
Sample
name
97a'
2379
3400 a
3400
12046
3493
3794
SW480 a
SW480
APC
genotype
apc+/apc+
apc+/apc+
apc~/apc+
apcm/apc+
apc~/apc+
apc'/apc+
apc~/apc+
apcm/apcm or apcm/apcm/apcm or apcm/-
Position of
APC
mutation
No. of
blue
colonies
No. of
white
colonies
No. of
light blue
colonies
4015
4015
3183
3926
2805
4017
4017
388
91/91
188
74/74
33/33
64/64
76
4
3/3
18
10/3
25
23/10
20/16
16/7
2
14
19/7
16
3/10
257
108/121
44/48
73/82
94
332
224/236
a DNA ligation was set up in a 1:2 vector:insert ratio.
shown). However, 4% of colonies derived from DNA of a normal
subject also were intermediate blue, implying that mutations were also
present in these inserts, most likely generated by PCR.
The five DNAs used for this assay had been characterized previously using other methods of mutation detection. Each DNA sample
was taken from an individual or tumor cell line with a different APC
genotype: apc+/apc +, apcm/apc § and apcm/apcm (or apcm/apc-). All
five carried unique APC mutations within the region of the gene
amplified by the primers.
Each of these five different germline or tumor cell line APC mutations was detected with the PCR/cloning strategy presented here.
Table 1 summarizes the results. The colony colors and their ratios for
three samples are shown in Fig. 2. DNA sequencing of colonies
derived from several of these assays gave the expected sequences in
every case (data not shown).
Discussion
or stability of the chimeric protein could be important. Indeed, the
omission of IPTG from the plating steps of this assay was indicated by
initial experiments suggesting that overexpression of the cloned gene
product, when induced by IPTG, resulted in small, slow-growing
colonies. This observation implies toxicity either from the high expression of the chimeric protein or from the vector in the Sure strain
of bacteria.
Heterogeneity in colony color is observed with the samples tested
here. In some platings, the intensity of the blue color obtained from
normal DNA inserts varied, especially in comparison to the lighter
blue color obtained from the mutant inserts. Although it was not
difficult to differentiate colors among colonies on a single plate,
comparisons from one plate to another were difficult to interpret.
Colors obtained from samples with frameshifts in APC showed clear
differences between the blue and the intermediate blue colonies. Colors obtained from samples with premature stop codons, however, gave
a less obvious, although still distinguishable, difference in color. The
type of stop codon present also slightly affected the colony colors
obtained: ochres (UAG) gave a clearer difference in the two colony
colors than did amber (UAA) codons. We believe that this observation
reflects the ability of bacteria in general to read through some stop
codons more efficiently than others. In general, ochre codons can be
read through more efficiently than amber codons, although in this
instance, this ability is tempered by the supE44 genotype of the Sure
strain. This gene is a nonsense suppressor that allows for about 1020% of ambers to be read as glutamines. Finally, UGA is the least
efficient of the termination codons (18). Work is in progress to test
other bacterial strains with different genotypes to optimize color differences. Surprisingly, other strains tested (su1675 and HB101) that
do not have the supE44 mutation do not generate results that as are
clear as those using Sure.
The genomic structure of the APC gene is unusual, in that one of its
exons is 6.5 kilobases. This size permits the majority of the open
reading frame to be screened using genomic DNA. Moreover, the
majority of germline mutations identified to date have occurred in this
exon (2-11). The remainder of the gene is composed of at least 19
exons that are alternatively spliced (12, 17). 5 Therefore, the remainder
of the open reading frame will be most effectively screened through
complementary DNA. This can be accomplished with one or two sets
of primers, depending on the splice forms that are targeted for analysis. 6 Most human disease genes that are candidates screening with the
method reported here, i.e., those known to carry a significant proportion of nonsense or frameshift mutations, will likewise be screened
most efficiently by using complementary DNA.
Mutational analyses of colorectal adenomas and carcinomas at the
APC locus have shown that most, if not all, tumors carry at least one
nonsense or frameshift APC mutation (3, 19-21). Many of the tumors
analyzed carry two APC mutations or show a loss of heterozygosity
affecting the APC region of chromosome 5 (19-21). 7 These observations underscore the potential effectiveness of the mutation screening
method presented here for the detection of APC mutations in colorectal adenomas and carcinomas, as well as in APC patients. These
same mutational analyses of tumors also have suggested that over
70% of APC mutations occur within a 3-kilobase region of exon 15.
The fact that this region is contained within the 6.5-kilobase contiguous genomic sequence of open reading frame suggests that genomic
DNA can be used as a PCR template in the assay reported here.
The experiments presented here have shown that a specific segment
of the APC gene can be scanned efficiently and reliably for nonsense
and frameshift mutations with a colorimetric assay. Work is under way
This mutational screening technique detects germline mutations in
the APC gene in patients and their family members who may be at risk
for the inheritance of the same mutation. Detection of the mutations is
accomplished by a rapid colorimetric assay. If the result suggests that
a nonsense or frameshift mutation is present, repetition of the same
assay with shorter stretches of the gene would localize the mutation
more specifically. Direct sequencing of the PCR product would identify the exact mutation.
The efficiency of this assay is determined by the size of DNA
segments that can be screened. More conventional methods of mutation screening, such as single strand conformation polymorphism
analysis, denaturing gradient gel electrophoresis, temperature gradient
gel electrophoresis, direct sequencing, or RNase protection assays, are
all labor-intensive, gel-based assays that are normally reliable for
DNA segments no longer than about 500 nucleotides. Other methods
that are not gel based, such as allele-specific PCR and the oligoligation assay, are directed only toward the identification of known
mutations and are not applicable for detection of unknown mutations
(8, 15). In APC screening, for example, these methods would be
inefficient, as none of the mutations identified to date has been found
at a frequency of more than 1 in 10. A functional assay for the
detection of p53 mutations in Li-Fraumeni patients has been described; however, in this case, it is the function of the gene product
itself that is assayed and thus not generally applicable (16).
Although the DNA segment length chosen for this assay is under 2
kilobases, the size of a fragment that can be screened depends upon a
combination of the ability of PCR to generate larger fragments of
DNA and the ability of/3-galactosidase to be expressed and to function appropriately with a second, uncharacterized protein domain attached. Although the function of/3-galactosidase is not usually affected by the gene product attached, factors such as solubility, toxicity,
5583
5 R. White, unpublished data.
6 L. Spirio, unpublished data.
7 L. Varesco, unpublished observations.
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SCREEN FOR APC MUTATIONS
to i m p r o v e the c o l o r i m e t r i c d e t e r m i n a t i o n o f the assay and to e x t e n d
this m e t h o d to include the r e m a i n d e r o f the A P C gene, b y u s i n g other
p r i m e r pairs and b y m u l t i p l e x i n g the P C R and c l o n i n g reactions. W e
h o p e that the m e t h o d can b e applied in clinical situations for the
a c c u r a t e and rapid d i a g n o s i s o f A P C .
Acknowledgments
We would like to thank Richard Cawthon, Tim Galitski, and Thrr~se Touhy
for helpful discussions and Ruth Foltz for critical reading of the manuscript.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer
Research.
A Rapid Screening Method to Detect Nonsense and
Frameshift Mutations: Identification of Disease-causing APC
Alleles
Liliana Varesco, Joanna Groden, Lisa Spirio, et al.
Cancer Res 1993;53:5581-5584.
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