Virus Genes (2008) 37:22–30
DOI 10.1007/s11262-008-0239-8
A non-radioactive PCR-SSCP analysis allows to distinguish
between HPV 16 European and Asian-American variants in
squamous cell carcinomas of the uterine cervix in Colombia
Pablo Moreno-Acosta Æ Mónica Molano Æ Antonio Huertas Æ
Myriam Sánchez de Gómez Æ Alfredo Romero Æ Mauricio González Æ
Marı́a Mercedes Bravo Æ Alejandro Garcı́a-Carrancá
Received: 21 November 2007 / Accepted: 2 May 2008 / Published online: 30 May 2008
Ó Springer Science+Business Media, LLC 2008
Abstract Human Papillomavirus type 16 (HPV 16) DNA
is regularly found in around 50% of all cervical carcinomas.
Variants of this type have been found associated with dif-
ferent risks for cervical cancer development. Presence of
HPV 16 variants in Colombia has not been previously
reported. The aims of this study were to assess the feasibility
of non-radioactive PCR-SSCP (polymerase chain reaction
single-strand conformation polymorphism) analysis for
determination of variability of ORF of E6, variability in the
enhancer sequence of the LCR, and for establishment of the
distribution of HPV 16 variants in invasive squamous cell
carcinoma of the uterine cervix in Colombian women.
Biopsies from 59 patients at the Instituto Nacional de Can-
cerologı́a (INC) in Bogotá (Colombia) were collected. HPV
detection was performed using universal primers. HPV 16
variants were detected by non-radioactive single-stranded
P. Moreno-Acosta (&)
M. Molano
A. Huertas

M. Mercedes Bravo
Grupo de Investigación en Biologı́a del Cáncer, Instituto
Nacional de Cancerologı́a, Calle 1a. No. 9-85, Bogota, D. C.,
Colombia
e-mail: dajup63@yahoo.com; pmoreno@cancer.gov.co
M. Molano
e-mail: mmolano@cancer.gov.co
A. Huertas
e-mail: antonio27col@yahoo.com
M. Mercedes Bravo
e-mail: mbravo@cancer.gov.co
M. S. de Gómez
Laboratorio de Hormonas, Departamento de Quı́mica,
Universidad Nacional de Colombia, Carrera 30 Calle 45, Bogota,
D. C., Colombia
e-mail: mysanchezd@unal.edu.co
123
conformational polymorphism (SSCP) analysis and direct
sequencing. HPV 16 was detected in 57.6% of the tumors.
The European branch was identified in 88.2% of the samples
with the E-G350 class being the most prevalent variant
(41.1%). The Asian-American branch was identified in 8.8%
of the samples. Within this group it was possible to distin-
guish between c and a classes. It was not possible to
determine the branch in 2.9% of the cases. A nucleotide
transition (G to A) at position 7521 was the most prevalent
variation (80%) found in the enhancer sequence of the LCR
region. Conclusion: A non-radioactive PCR-SSCP analysis
allowed us to distinguish between European and Asian-
American branches of HPV 16, and to distinguish among
classes in squamous cell carcinomas of the uterine cervix in
Colombia. This method is an excellent alternative that can be
used as a screening tool for identification of HPV 16 variants.
A. Romero
Patologı́a, Instituto Nacional de Cancerologı́a, Calle 1a. No 9-85,
Bogota, D. C., Colombia
e-mail: aromero@cancer.gov.co
M. González
Clı́nica de Ginecologı́a, Instituto Nacional de Cancerologı́a,
Calle 1a. No 9-85, Bogota, D. C., Colombia
e-mail: mgonzalezc@cancer.gov.co
A. Garcı́a-Carrancá
Unidad de Investigación Biomédica en Cáncer, Instituto de
Investigaciones Biomédicas, Universidad Nacional Autónoma de
México – Instituto Nacional de Cancerologı́a, Secretarı́a de
Salud, Mexico, D. F., Mexico
e-mail: carranca@correo.biomedicas.unam.mx
Virus Genes (2008) 37:22–30
Keywords HPV 16
Variants
PCR-SSCP

Cervical cancer
Colombia
Introduction
In Colombia, cervical cancer constitutes the first cause of
death among women of reproductive age [1, 2]. Sufficient
evidence exists to show that persistent infections with
human papillomavirus (HPV) are necessary for the devel-
opment of invasive cervical cancer [3]. HPV type 16 (HPV
16) is by and large the most frequently detected genotype
in cervical cancer where it accounts for approximately
53.5% of all tumors [4–7].
Variants have been defined as HPV with less than 2%
nucleotide differences in coding regions and up to 5%
nucleotide differences in the LCR.
The variants of each HPV type form phylogenetic trees,
and variants from specific branches are often unique to
specific ethnic groups [8–13]. A growing quantity of epi-
demiological, etiological, and molecular data suggests that
variants of the same HPV type are biologically distinct and
may confer differential pathogenic risks [8, 14, 15].
In many studies, non-European variants of HPV 16 have
been found associated with an increased risk for develop-
ment of cervical cancer, but other studies have not shown
this relation [14, 16–19].
Sequence variations within the LCR may have an impact
on the oncogenic potential of the virus [20–22]. In HPV 16,
transcription of E6 and E7 viral oncogenes is controlled by
the P97 promoter (located at the 30 end of the LCR) and by
an enhancer that contains several binding sites for cellular
and viral transcription factors [23]. The activity of the pro-
moter and enhancer are regulated by both viral and cellular
proteins, which mediate their transcriptional activity
through interaction with specific protein-binding sites
within the LCR. Cellular transcription factors such as YY1
can participate in the down regulation of the HPV 16 pro-
moter. Some changes in YY1 binding sites may enhance
potential for the expression of viral oncoproteins [24, 25].
The E6 region of HPV 16 variants exhibits changes in
functional or antigenic domains that can alter the biological
or immunogenic properties of the encoded protein [19, 26].
A change at position 350 (T to G substitution; L83V)
within the E6 ORF has been shown to lead to important
antigenic changes [19] which in many studies have been
associated with viral persistence and progression of pre-
malignant cervical disease [27–32].
The aim of this study was to assess the feasibility of a
non-radioactive PCR-SSCP analysis in the determination
of variability of ORF of E6 and the enhancer sequence of
the LCR, and establishment of the distribution of HPV 16
variants in invasive squamous cell carcinoma of the uterine
cervix in Colombian women.
23
Methods
Clinical samples
Patients with a cytological diagnosis of invasive cervical
cancer were referred to the Gynecology Clinic at the In-
stituto Nacional de Cancerologı́a (INC) in Bogotá,
Colombia. Biopsies were collected from 59 patients.
Clinical stages ranged between IIB and IIIB (FIGO). The
mean age of the patients was 51 years (range 30–76). This
study received official institutional and ethical approvals.
The samples were frozen at -70°C and used to analyze
HPV DNA by PCR assays.
PCR assays
To verify the quality of the target DNA, PCR analysis of
the b-globin gene was performed using PCO3 and PCO5
primers [33]. The presence of HPV DNA was detected
using consensus primers (GP5+/GP6+) [34]. Three dilu-
tion series of SiHa cells and plasmid DNA were used as
positive controls for HPV 16 (pBR322 HPV 16). The
central segment of the HPV16 LCR (nucleotides 7409–
7891) was amplified as previously described [35]. Twenty-
seven amplification cycles were carried out using a Perkin
Elmer 9600 (USA) thermocycler. The products were then
analyzed by direct sequencing.
Considering the sensitivity of the SSCP and variations in
sequence of the reported E6 gene, two internal regions were
selected to the ORF of E6: nucleotides 104–227 were
amplified to identify changes at 145 position to distinguish
between European variants and non-European variants (e.g.
Asian-American variants); these nucleotides were amplified
using sense 50 -ATGTTTCAGGACCCACAGGAGCGA-30
and antisense 50 -CCTCACGTCGCAGTAACTGT-30 prim-
ers. PCR was carried out in a volume of 50 ll containing 50
mM KCl, 20 mM Tris–HCl pH 8.3, 0.2 mM dNTPs, 2 mM
MgCl2, 1 U of Taq Polymerase (GIBCO BRL), and 0.25 lM
of each primer. Thirty-eight amplification cycles were car-
ried out by using a Perkin Elmer 9600 (USA) thermocycler
[36]. The expected size of the amplification product was 123
base pairs. Also nucleotides 314–490 were amplified to
identify a change at 350 position to distinguish between E-
G350 class and E-T350 class. The expected size of the
amplification product was 176 base pairs. Three dilution
series of HPV 16 reference clone (pBR322 HPV 16r), SiHa
and CaSki cells lines DNA were used as positive controls,
PCR mix without DNA and water were used as negative
controls.
Finally the ORF of E6 (nucleotides 63–559) was
amplified to detect changes in sequence not analyzed
through SSCP.
123
24
The E6 ORF was amplified by using sense E6F1 50 -
AAACTAAGGGCGTAACCG-30 and antisense E6R1 50 -
TGTAGGTGTATCTCCATGC-30 primers. PCR was car-
ried out in a volume of 50 ll containing 50 mM KCl, 20
mM Tris–HCl pH 8.3, 0.2 lM dNTPs, 2.5 mM MgCl2, 1.25
U of Taq Polymerase (GIBCO BRL), and 0.5 lM of each
primer. Forty cycles of amplification were carried out.
Non-radioactive SSCP analysis
HPV 16 positive samples were identified by means of non-
radioactive PCR-SSCP analysis [37, 38]. In brief, 3 ll of
amplified target were mixed with denaturing solution and
running in a 12.5% non-denaturing polyacrylamide gel for
4.5 h at 400 V with cooling at 8°C.
Analysis of E6 HPV 16 variants was done also using a
non-radioactive PCR-SSCP with similar conditions to
those described above, except that running was at 400 V,
overnight, and at 8°C. For this analysis, dilution series of
SiHa DNA were used as a positive control of European
variants, dilution series of CaSki DNA were used as a
positive control for A131G variation [39], and specimens
previously identified and quantified in our laboratory were
used as positive controls for A145T variation.
CaSki cells DNA was used as a positive control for
detection of the G350 variation, and the pBR322 HPV 16
was used as a reference control [39]. The conditions for
SSCP were the same to those described for E6 SSCP.
DNA sequencing
Amplified DNA was purified by spinning column purifi-
cation (GFX Amersham Pharmacia Biotech). Cycle
sequencing was performed by using 7-Deaza-dGTP-Cy5.5
Dye Primer Cycle Sequencing Kit Protocol (Visible
Genetics Inc., Toronto) according to the manufacturer and
run on an automated DNA sequencer (SEQ 4 9 4, Amer-
sham Pharmacia Biotech). Sequences of both sense and
antisense strands of the PCR products were generated. The
central fragment of LCR (nucleotide 7409–7891) and ORF
of E6 (nucleotide 63–559) were amplified and sequenced.
Sensitivity and specificity assays
During the standardization procedure of the different
PCRs, SSCP analysis, and direct sequence we made dif-
ferent sensitivity and specificity assays. For the sensitivity
analysis we made dilution series (100 ng–10 fg) of HPV 16
reference clone (pBR322 HPV 16r), SiHa and CaSki cell
lines DNA, respectively. For the specificity assays we used
DNA of different HPV types, C. trachomatis DNA, human
DNA, and H. pylori DNA.
123
Virus Genes (2008) 37:22–30
In addition, reproducibility of the PCR-SSCP analysis
was evaluated by the generation of the same SSCP pattern
for the E6 fragments in assays done in triplicate of the
controls. During the experimental analysis different sam-
ples were also analyzed in duplicate, again confirming the
reproducibility of the results.
System designed for HPV16 branch/class
The nucleotide positions of the HPV 16 genome were
numbered according to the reference sequence of the HPV
16 genome (HPV 16r) [40]. Variation designation was
performed according to Yamada et al. [11].
Variant designation was done according to Huertas et al.
(paper submitted). Briefly, this description proposes an
updated and unified criterion for clustering and nomen-
clature of HPV16 E6 molecular variants based on
systematic revision of comparative analysis of previously
published nucleotide sequences.
Results
Sensitivity and specificity assays
These assays showed that the level of sensitivity among
different PCRs ranged from 1 ng to 10 pg depending on the
amplified fragment. Sensitivities of the SSCP assay for
small PCR fragments reached 10 pg. However by direct
sequencing we were able to detect up to 100 pg of DNA.
Specificity analysis of the different PCRs showed no cross
reactions with different HPV types (18, 31, 33, and 58) and
also no reaction with others DNAs such as C. trachomatis,
human DNA, and H. pylori. The SSCP analysis was real-
ized under standardized conditions (temperature 8°C,
constant voltage, composition of the load mixture), which
avoided appearance of non-specific additional bands.
Molecular variants in the ORF E6 of HPV 16 among
tumors in Colombia
HPV 16 was detected in 57.6% of the tumors (34/59).
Frequencies of HPV 16 variants detected are shown in
Table 1: European branch was identified in 88.2% of the
samples, with the E-G350 class being the most prevalent
(41.1%). Asian-American branch was identified in 8.8% of
the samples, among which it was possible to distinguish
between c and a classes. In 2.9% of the samples it was not
possible to determine the branch. Variation G350 was the
most prevalent (52.8%) in our study. To distinguish dif-
ferent HPV 16 variants, we set out to establish patterns of
different variants of HPV 16 in a non-radioactive SSCP
analysis. This was followed by direct sequencing.
Virus Genes (2008) 37:22–30
Table 1 HPV 16 variants frequencies in cervical carcinoma patients
Frequency %
Cancer patients
n = 34
Branch E 30 (88.2)
Class E-T350 12 (35.2)
Class E-G350a 14 (41.1)
Class E-nd 4 (11.7)
Branch AA 3 (8.8)
Class AA-a 2 (5.8)
Class AA-c 1 (2.9)
Branch nd (NE) 1 (2.9)
HPV 16 branch/class designation as described in the methods section.
E: European variant; AA: Asian-American variant; NE: non-Euro-
pean variant; nd: Not determined variants
a
Included Subclass E-G131/G350
We established a pattern in SSCP analysis that allows us
to distinguish between European and non-European vari-
ants (e.g. Asian-American variants) of HPV type 16
(Fig. 1a, b). Representative SSCP patterns of European
variants (lanes 1, 3, 5, 6, 7, 8, 9) and non-European variants
(lanes 2, 4, and 10) from samples included in the study are
shown in Fig. 1c. Sample 61 (lane 1) exhibited a pattern
similar to European variants, but not identical. With
sequence analysis we were able to detect a G-to-A sub-
stitution at position 131 (subclass E-G131).
Analysis of variations in the segment spanning from
nucleotides 314 to 490 of the E6 gene showed different
SSCP patterns between the reference clone of HPV 16
A B
ds DNA
E G145 T183
1 2 3 4
C
ds DNA
Fig. 1 Distinction between European and non-European variants of
HPV 16 by means of SSCP and sequence analysis. (a) Representative
SSCP patterns of European variants (E); sequence analysis of
nucleotides G145 and T183. (b) SSCP patterns for non-European
variants (NE); sequence analysis of nucleotide 145, with presence of
thymidine (T), and of nucleotide 183, with presence of guanine (G).
(c) Representative SSCP patterns of European variants (lanes
25
(pBR322 HPV 16r—T350, Fig. 2a) and CaSki cell line
(G350, Fig. 2b). This allowed us to specifically detect
changes at 350. Representative SSCP patterns of T350
(Fig. 2c, lanes 2 and 3) and G350 (Fig. 2c, lanes 1, 4, and
5) from samples included in the study are shown.
In Table 2 variations observed in the ORF E6 for the
European branch are shown. Three groups distinguish
themselves. One corresponds to samples that do not present
any change in sequence and which correspond to European
variant class E-T350 [11]. Another only presents change at
G350 and corresponds to European variant class E-G350
[11]. The third group had changes at position 350 which
could not be precisely determined. We labeled this group
European variant non-determined.
Another SSCP pattern that we were able to identify was
the Asian-American branch. Within this branch we were able
to distinguish between class a and class c of type 16 Asian-
American branch variants (Fig. 3a, b). This was confirmed
by sequencing (Class a showed a T at position 183 (Fig. 3a),
and class c showed a G at position 183 (Fig. 3b)).
It was not possible to determine the branch, class, or
subclass for 2.9% of the samples. These were denoted as
not determined (nd) variants; however, with the SSCP
analysis they showed a pattern similar to the non-European
variants (sample 4, Fig. 1c, lane 10).
Molecular variants in the enhancer sequence
of the LCR of HPV 16
Of the 34 samples which were positive for HPV 16, we were
able to amplify the enhancer sequence of the LCR
NE T145 G183
5 6 7 8 9 10
1,3,5,6,7,8,9 (samples 61,26,22,12,10,8, and 6, respectively)) and
non-European variants (lanes 2,4, and 10 (samples 29, 24, and 4,
respectively)) from samples included in the study. The arrow
indicates double-strand DNA (ds DNA). Differences observed in
SSCP patterns between European and non-European variants, which
correspond to nucleotide changes, were confirmed by direct
sequencing
123
26
A B
ds DNA
T350 T350
1 2
C
ds DNA
Fig. 2 Detection of the G350 change through SSCP and sequence
analysis. (a) SSCP patterns for E6 without change at nucleotide 350
from DNA of pBR322 HPV 16 reference clone and sequence
analysis. (b) SSCP pattern for E6 from DNA of the CaSki cellular line
shows an additional band (white arrow) of higher intensity that
represents a change at position 350 and sequence analysis showing
(nucleotides 7409–7891) in twenty-one samples. In 15 of
them it was possible to detect variations by direct sequencing.
The samples predominantly carried changes in sequences of
YY1 binding sites, especially at nucleotide 7521 (G to A)
with a frequency of 80%. Other notable changes were at
nucleotides 7786 (C to T), 7485 (A to C), and 7469 (T to A).
Changes in sequences of TEF-1, GRE/2, and Oct-binding
sites were infrequent (13.3%, 6.6%, and 6.6%, respectively).
Five molecular variants were detected. Of these, one is a
non-European variant, while four belong to the European
branch. The non-European variant had the following
nucleotide changes: A to C at 7485 nt, G to A at 7489 nt, G to
A at 7521 nt, C to T at 7669 nt, C to A at 7689 nt, A to C at
7729 nt, C to T at 7764, and C to T at 7786 nt. This variant
corresponds to the Asian-American variant class c detected
in the ORF E6. From the European variants, one had the
reference sequence, another had a change T to A at 7469 nt,
another had a change G to A at 7521 nt, and the other had a
change G to A at 7489 nt plus a 7521 change. All variants
found in the enhancer sequence of the LCR are described in
Table 2. Their frequencies are shown in Table 3.
Discussion
In the present study, HPV 16 was the most prevalent type
detected in women with invasive squamous cell carcino-
mas of the uterine cervix, stages IIB and IIIB (FIGO). This
is in accordance with the majority of studies which also
report HPV 16 to be the most frequent type detected in this
kind of carcinoma [3, 6, 36, 41].
123
Virus Genes (2008) 37:22–30
G350 G350
3 4 5
the change T to G at this position. (c) Representative SSCP patterns of
T350 (lanes 2 and 3 (samples 8 and 12, respectively) and variation
G350 (lanes 1,4, and 5, samples 6, 24, and 26, respectively) from
samples included in the study. Black arrow indicates double-strand
DNA (ds DNA)
We were able to characterize the genetic variability of
ORF of E6 and the enhancer sequence of LCR in tumors
from 34 Colombian women infected with HPV 16 and to
determine the distribution of HPV 16 variants.
The majority of Colombian women analyzed in our study
exhibited European variants of HPV type 16. Although
reports about the frequency of E6 variants in South and
Central America [11] had provided a general view of their
distribution, they had not given specific data about the fre-
quency of these variants in Colombia. Our analysis indicate
similar frequencies as those predicted and reported by
Yamada et al. [11] for European variants (88.2% vs. 76.8%,
respectively), for the E-350T prototype (35.2% vs. 24.6%,
respectively), for E-350G (41.1 % vs. 52.2%, respectively),
and for non-European variants, specifically Asian-American
variants (8.8% vs. 19.7%, respectively).
European variant E-G350 class was the most prevalent in
our study, and although little is known about the functional
consequences of sequence variations regarding E6 protein
activity, recent studies suggest that naturally occurring amino
acid variations in HPV 16 E6, such as G350 corresponding to
L83V, can alter protein activities that are important for its
carcinogenic potential [42]. In addition, L83V enhances
MAPK signaling and cooperative transformation with de-
regulated Notch1 signaling [28]. In four samples position 350
could not be defined, possibly due to a low viral load.
The analysis of the enhancer sequence of the LCR region
in our study showed that the most frequent nucleotide change
was at position 7521 (A to G), which is in agreement with the
majority of reports [35, 43]. This change was present in 80%
 Table 2 Nucleotide changes in the E6 and enhancer sequence of the LCR of different HPV 16 samples
Branch Class Subclass HPV 16r ORF E6 Predicted amino acid change LCR Samples
7 7 7 7 7 7 7 7 7 7
1 1 1 2 2 3 3 5 4 4 4 4 5 6 6 7 7 7
3 4 8 8 8 3 5 3 3 6 8 8 2 6 8 2 6 8
1 5 3 6 9 5 0 2 6 9 5 9 1 9 9 9 4 6
A G T T A C T A G T A G G C C A C C

E T 350 /r – – – – – – – – – – – – – – – – – – 46, HPV-16r

Virus Genes (2008) 37:22–30

– – – – – – – – — nd 22, 27, 31, 57

– – – – – – – – — – A – – – – – – – – 52
– – – – – – – – — – – – – A – – – – – 7, 9, 12
– – – – – – – – — – – – – A – – – – – 8
– – – – – – – – — – – – – A – – – – – 41
– – – – – – – – — – – – A – – – – D SiHa
nd – – – – – – – — – – – – A – – – – – 10
G 350 /G 350 – – – – – – G – L83V – – – – A – – – – – 6, 40, 42, 53, 56
– – – – – – G – L83V nd 17, 19, 26, 36, 44
nd – – – – G – L83V nd 16, 25, 47
/G 131 /G 350 G – – – – – G – R10G, L83V – – – A A – – – – – 61
G – – – – – G – R10G, L83V – – – – A – – – – – CaSki
nd nd – – – – – – nd – nd nd 15, 18, 45, 60
AA a /r – T – a g T G g Q14H, H78Y, L83V nd 24, 54
c /r – T G a g T G g Q14H, 127R, H78Y, L83V – – C A A T A C T T 29
nd (NE) nd nd – T – – – – G – Q14H, L83V nd 4
Transcription factor binding site TFF- YY1 YY1 TEF- YY1
1 1
YY1 GRE2
Oct-1
DNA sequence analysis of HPV 16 variants from nucleotide position 559 to 63 of E6 gene by SSCP analysis and by direct sequencing of polymerase chain reaction products and in the enhancer
sequence of the LCR from nucleotide position 7893–7409 by direct sequencing of polymerase chain reaction products. The nucleotide positions of the HPV 16 genome were numbered
according to the reference sequence of the HPV 16 genome (HPV 16r) [40], variation designation was performed according to Yamada et al. [10] and designation of branches or class and
subclasses according as described in methods section. The variations observed in the E6 open reading frame (ORF) for the European branch are shown trough of three groups that are indicated
from above to below: The first group corresponding to samples that do not present any change in sequence and that corresponds to the European variant class E-T350, the second group
corresponding to samples that only presents change at G350 and corresponds to European variant class E-G350 and the third group corresponding to samples that had changes at position 350
which could not be precisely determined. We labeled this group European variant not determined. For the Asian-American branch established in the ORF E6 are showed two groups: The first
group corresponding to samples that were designed as class a and a second group corresponding to samples that were designed as class c. The samples in which was not possible to determine the
branch, the class or the subclass through of the analysis of the ORF E6, were denoted as not determined variants. Nucleotide changes involving know transcription factor binding sites are
indicated in the bottom alignment. TEF-1: Transcriptional Enhancer Factor-1; YY-1: Yin-Yang Factor 1; GRE: Glucocorticoid Response Element; Oct-1: Octamer Binding Factor-1. Nucleotide
positions are indicated numerically and vertically and letters refer to the amino acid sequence of the reference clone (HPV16r). A letter immediately following a dash and preceding a nucleotide
position number represents the variation at the specific nucleotide designated compared with the reference sequence. Capital letters indicate alterations in the ORF E6 that result in amino acid
changes, while lower case letters indicate silent mutations. ‘‘–’’ Indicates that the reference sequence was observed at that position. nd: Not determined. D: deletion (nucleotides 7758–7794 in
27

123
the enhancer sequence of the LCR). E: European variant; AA: Asian-American variant; NE: non-European variant
28 Virus Genes (2008) 37:22–30

Fig. 3 Distinction between A B

classes of the Asian-American
(AA) variants by means of
SSCP and sequence analysis. (a)
Representative SSCP patterns
for variant AA-a and sequence
analysis for the nucleotide
T183. (b) SSCP patterns for
variant AA-c and sequence
analysis showing the change T
to G at 183 position. The arrow
indicates double-strand DNA
(ds DNA)
ds DNA

AAa T183 AAc G183

Table 3 Frequency of nucleotide changes found within the enhancer 7781–7790 of the LCR, that are next to a YY1 binding site,
sequence of the HPV 16 LCR can increase promoter activity from 2 to 4 times when
LCR Frequency % compared with the reference LCR, thus supporting the
nt. 7409 at nt. 7803 n = 15 hypothesis that mutations in a site next to YY1 are asso-
7521 A–G 12 80.0
ciated functionally with the development of cervical
cancer. Indeed, studies by O’Connor et al. [46], have
7521 A–G 1 6.6
indicated the importance of YY1 in the regulation of HPV
7489 A–G
16 E6 and E7 gene expression.
7521 A–G 1 6.6
In our study, the enhancer sequence of the LCR could
7485 C–A
not be amplified in 13 samples, possibly due to loss of
7489 A–G
sequence (deletion) in this region [44] and low number of
7669 T–C
viral copies.
7689 A–C
It is well known that the sensitivity of SSCP analysis is
7729 C–A
based on characteristics of polymerase chain reaction
7764 T–C
products including: the size of the fragment (optimal size is
7786 T–C
less than 300 base pairs); sequence composition of the
7469 A–T 1 6.6 fragment (low number of Guanine and Cytosine (GC)), and
The frequency of variation for the LCR central fragment was esti- utilization of DNA minimal quantity. For our E6 PCR-
mated from the variations in sequence of 15 samples. The variations SSCP analysis we amplified products of 123 and 176 pb,
found in the enhancer sequence of the LCR (nucleotides 7409–7893)
which are the ideal size for the detection of base substi-
are located in the first column. The most common nucleotide change
was found in position 7521 tution [37]. The sequence composition of the fragment
amplified also had a low number of GC and we put only 3
ll of PCR product obtained for 10 ll of pre-treated crude
cell suspension. Therefore, a standardized, non-radioactive
of HPV 16 variants in this study. This and other mutations PCR-SSCP analysis allows detection of fragments that
seem to alter the binding of the transcription factor YY1 and have base substitutions (sequence variation confirmed by
sometimes are considered functional [35, 43–45]. For direct sequencing), and all of these base substitutions result
example, in one case (case 29) we found a c-class Asian- in detectable shifts in fragment mobility. The presence of
American variant that, in addition to the change at nt. one or more bands of greater intensity allowed us to detect
(nucleotide) 7521(A to G), showed changes at nt. 7485 (A to nucleotide variation (specifically to detect the G350 vari-
C), nt. 7489 (G to A), and nt. 7786 (C to T). According to Park ation in the E6 gene), to distinguish between variants, to
et al. [43], these are mutations placed in YY1 binding sites. In differentiate between European and Asian-American vari-
the same way, changes at nt. 7436 (case 10), according to ants, and to differentiate between classes.
Schmidt et al., 2001, and changes at nt. 7489 (case 61), Although methodologically we did not propose to
according to Park et al. [43], will be localized within this site. independently determine variants of E6 of HPV 16 by
Previous reports [43, 46], suggested that mutations means of SSCP and sequencing, we found that in some
at nucleotides 7481–7489 and mutations at nucleotides samples (s16, s19, s22, s25, s26, s31, s56), the class of

123
Virus Genes (2008) 37:22–30
variant was defined by SSCP. The determination of
nucleotide changes in these samples by using sequencing
was not possible. This was probably due to the higher
sensitivity of the PCR-SSCP compared with the direct
sequence, also to problems inherent to the sample and/or
low viral copies. These results allow us to affirm that the
SSCP was more sensitive than was direct sequencing.
To conclude, a non-radioactive PCR-SSCP analysis with
high reproducibility allowed us to distinguish between HPV
16 European and Asian-American branches and classes in
squamous cell carcinomas of the uterine cervix in Colombia.
This method is an excellent alternative that can be used as a
screening tool for identification of HPV 16 variants.
The description and report of frequency of HPV 16
variants contribute to our understanding of the role that
these variants play as risk factors for invasive squamous
cell carcinoma of the uterine cervix in our population.
Acknowledgments We thank Bernardo Avellaneda for his help in
the preparation of section biopsies. We also acknowledge to Marcela
Lizano for critical reading of the manuscript. This work received
financial support from the Instituto Nacional de Cancerologı́a
(Colombia) through the investment project ‘‘Use of Cold Probes in
the Study of the Relation between HPV and Cancer of the Uterine
Cervix’’ (Code 4103038-1).
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