Arch Virol (2014) 159:3305–3320 DOI 10.1007/s00705-014-2199-8

ORIGINAL ARTICLE

Molecular variants of human papilloma virus 16 E2, E4, E5, E6 and E7 genes associated with cervical neoplasia in Romanian patients Adriana Plesa • Gabriela Anton • Iulia V. Iancu • Carmen C. Diaconu Irina Huica • Anca D. Stanescu • Demetra Socolov • Elena Nistor • Elena Popa • Mihai Stoian • Anca Botezatu

•

Received: 3 March 2014 / Accepted: 30 July 2014 / Published online: 21 August 2014 Ó Springer-Verlag Wien 2014

Abstract The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p \ 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia

A. Plesa (&)  G. Anton  I. V. Iancu  C. C. Diaconu  I. Huica  M. Stoian  A. Botezatu Molecular Virology Department, Stefan S. Nicolau Institute of Virology, 285 Mihai Bravu Avenue, Sector 3, 030304, PO 77, PO Box 201, Bucharest, Romania e-mail: [email protected] A. D. Stanescu  E. Nistor  E. Popa ‘‘Bucur’’ Gynecologic Hospital, Bucharest, Romania D. Socolov ‘‘Grigore T. Popa’’ University of Medicine and Pharmacy, Iassy, Romania

and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis.

Introduction Of the over 150 different human Papillomavirus (HPV) types that can infect humans, 15 are associated with anogenital malignancy [1]. Romania has one of the highest incidences in Europe (31/100,000 per year), with the mortality rate in 2008 being 18.3/100,000 per year [2]. According to Apostol et al. [3], the standardized incidence of cervical cancer varies across the Romanian regions. For Moldavia (Iassy), the crude incidence rate (CIR) is 31.31, and the crude mortality rate (CMR) is 16.69, which is significantly higher than in the Bucharest region (17.8 and 12.35, respectively) [3, 4]. Although the high prevalence of HPV16 infection in precancerous and cancerous cervical lesions confirms its oncogenic potential, different HPV16 variants seem to be responsible for both CINIII and invasive cancer development. HPVs are double-stranded DNA viruses that use the DNA polymerase proofreading ability of the host cell and therefore mutate very slowly [5–7]. Factors that favor a small proportion of HPV16 infections to progress to cancer are still poorly understood, but several studies have reported sequence variations in early HPV genes, suggesting their contribution to carcinogenesis [8–13]. Different HPV16 variants induce biochemical and biological effects [14–17] that may affect virus assembly,

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pathogenicity, immunologic response, immortalization activity and transcription regulation [18–21]. There is a strong correlation between specific HPV16 variants and persistent viral infection, followed by development of malignant lesions [17, 22]. Moreover, HPV16 variants show different geographical distributions and oncogenic potential [23]. It is considered that a 2 to 10 % difference in the genome sequence defines a subtype, while less than 2 %, a variant [24]. Recently, major variant lineages are defined by an approximately 1.0 % difference between full genomes of the same HPV type, and 0.5 to 0.9 % differences define sublineages [5, 25]. Based on phylogenetic analysis of long control region HPV16 (LCR) variants, Ho [26] identified five major variant lineages, namely, EuropeanGerman (EG), Asian (As), Asian-American (AA), and two African lineages, African-1 and African-2 (AFR1 and AFR2). More recently, a new lineage has been added: North American 1 (NA1) [27]. Many studies suggest that non-European HPV16 variants are more oncogenic than EG [28]. Different HPV16 variants might constitute markers for onset and progression of cervical cancer and could partially explain why some lesions progress to cancer while others do not. For epidemiological studies, the most interesting variants are those of E6, E7 and more recently, of the E5 oncogene. Although E6 of HPV16 (nucleotide positions 83-144; 146-285; 351-526) is highly conserved [29], some regions have been found to show many variations. Recent studies have focused on sequence variation of the 50 end of the E1 and E6 genes from samples that harbored this genetic alteration [30]. As for the proteins coded by those oncogenes, the HPV16 E7 protein is highly conserved in vivo, and its tertiary structure does not tolerate amino acid substitutions [31]. Nevertheless, several studies have shown a range variation in the conserved region of E7 protein that correlates with cervical cancer [32, 33]. The HPV16 E5 protein is also an important mediator of oncogenic transformation [34]. During viral infection, E5 is expressed in early stages of neoplastic transformation and viral protein found in the membranous compartment of the cells increases the half-life of EGFR (epidermal growth factor receptor) [35, 36]. Effects of HPV16 E5 have been investigated in murine 3T3 cells upon transcription, cell cycling and cell growth, and it was found that E5 enhances the immortalization potential of E7 and, in association with E7, stimulates proliferation in vivo [37]. Infections with a combination of all HPV16 variants are associated with an increased risk of cervical cancer, in contrast with prototype HPV16 infection as well as with infection with only one of the variants [14]. Integration of the viral genome into host DNA, which is observed in cervical carcinomas, occurs in the E1/E2 ORFs and leads

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to disruption of E1 and/or E2 and to overexpression of E6/ E7 proteins, and thereby leads to deregulation of cell proliferation [38]. The E2 HPV16 protein consists of three distinct domains: the transactivation, hinge and DNA-binding domains. The hinge domain, which is apparently unstructured, is highly variable, whereas the structure and function of the other two domains are relatively conserved [39]. Sequence variations have been detected in all three domains. While E5, E6 and E7 variants have been fully characterized, data on E4 are still limited. The E4 protein plays an essential role during productive HPV infection, and most studies have focused on E4 gene variants in correlation with E5, E6 and E7 oncogenes. E4 variations have been detected in both N- and C-terminal domains of the viral protein. E4 gene is located entirely within the E2 gene and is expressed during the late phase of infection. Molecular evolutionary analysis of the E4 coding region has revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs [40]. This study was designed to analyze the sequence variations in the E4, E5, E6 and E7 HPV16 genes, as well as the sublineages present in cervical precancerous and cancerous lesions by comparing with sequences of HPV16 genomes from the GenBank database. We have also investigated specific variants of the HPV16 E2 gene and their involvement in disease progression. To our knowledge, this is the first study that aims to analyze the variants of HPV16 oncogenes in patients from two Romanian areas.

Materials and methods Specimen collection Three hundred thirty-eight paraffin-embedded cervical tissue samples were collected from women receiving gynecological service (Bucur Gynecologic Hospital, Bucharest – 168 samples; Grigore T. Popa University of Medicine and Pharmacy, Iassy –170 samples) for investigation of cervical lesions from 2010 to 2012. All of these patients were being examined for the first time. According to histopathological evaluations performed in accordance with WHO recommendation and CIN classification [2], the cervical diseases were categorized as follows: 104 samples with mild cervical intraepithelial neoplasia I (70 samples from Bucharest and 34 samples from Iassy), 89 samples with moderate cervical intraepithelial neoplasia II (41 samples from Bucharest and 48 samples from Iassy), 102 samples with severe cervical intraepithelial neoplasia III (37 samples from Bucharest and 65 samples from Iassy) and 43 samples with squamous cell carcinoma (20 samples

HPV16 variants associated with cervical neoplasia in Romania

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from Bucharest and 23 samples from Iassy). Written informed consent was obtained from all the patients, and the study was approved by the ethic committees of the gynecology clinics. The subjects (23-64 years old, median 40, mean ± SD, 40.67 ± 9.1) were divided into four groups according to the histological diagnosis for the two investigated areas. Bucharest: patients with CINI (median, 36; mean ± SD, 32.67 ± 7.57), CINII (median, 34.5; mean ± SD, 36.4 ± 9.05), CINIII (median, 39; mean ± SD, 40.75 ± 4.92), and SCC (median, 40; mean ± SD, 42.18 ± 8.06). Moldavia: patients with CINI (median, 33.5; mean ± SD, 34.8 ± 2.12), CINII (median, 36; mean ± SD, 33.67 ± 6.91), CINIII (median, 42; mean ± SD, 43.17 ± 7.32), and SCC (median, 46, mean ± SD, 47 ± 9.65). DNA isolation Five-lm-thick sections from each paraffin block were deparaffinized. A High Pure PCR Template Kit (Roche Molecular Biochemicals, Mannheim, Germany) was used to extract DNA according to the manufacturer’s procedures. One tube was processed according to the manufacturer’s protocol, using xylene treatment to remove paraffin as outlined in the DNeasy user manual (‘‘Purification of Total DNA from Animal Tissues’’ as found in the DNeasy Blood & Tissue Handbook, QIAGEN, Valencia, CA; July 2006). Briefly, the paraffin was removed by vortexing and a 10-minutes incubation with 1.2 mL xylene, followed by two washes with pure (200 proof) ethanol. The air-dried pellet was then incubated with 20 lL proteinase K and 180 lL ATL lysis buffer (from the DNeasy kit) for 16 hours in a heat block at 56 °C. The lysed emulsion was further purified using a DNeasy spin-column kit. DNA was finally recovered in a single elution step with 100 lL AE solution from the kit. The concentration and purity of each DNA sample were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Montchanin, DE).

Fig. 1 Agarose gel electrophoresis of PCR amplicons: 1, BenchTop 100 bp DNA Lader; 2, 3, and 6, samples from patients infected with HPV 16; 4, sample from a healthy patient; 5, negative control; 7, positive control; 8, *NTC. L1 HPV 16 gene, 450-bp amplicon; human b-globin gene, 268-bp amplicon. * NTC, no template control

HPV detection and genotyping Viral testing was performed with the Linear Array (LA) HPV Genotyping Test (Roche Molecular Biochemicals, Mannheim, Germany), according to manufacturer’s instructions. This test is a qualitative in vitro test for the detection of HPVs in clinical specimens. The LA HPV Genotyping Test detects 37 high- and low-risk human papillomavirus genotypes, including those considered a significant risk factor for HSIL progression to cervical cancer. The LA test utilizes amplification of target DNA by the polymerase chain reaction (PCR) and nucleic acid hybridization and is designed to amplify an approximately 450-bp sequence within the polymorphic L1 region of the

Fig. 2 Linear array HPV genotyping strip; hybridization signal for HPV 16 and internal control gene

HPV genome from 37 anogenital HPV DNA genotypes [6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9),

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Table 1 Primers and PCR cycling conditions for E2, E4, E5, E6 and E7 HPV16 genes HPV 16 gene

Sequence primers

Genomic position

Sequenced region

Annealing conditions

E2 transactivation domain

F-50 ATGGAGACTCTTTGCCAACG30

2755-2774

2755-3322

47 °C/60 s

R-50 ACCCGCATGAACTTCCCATA30

3302-3322

(567 bp)

3706-3726

3706-3864

E2 DNA binding

F-50 ACATGGCATTGGACAGGACA30 0

0

domain

R-5 CCAGTAATGTTGTGGATGCAGT3

3843-3864

(180 bp)

E2 hinge region/E4

F-5’TGGGAAGTTCATGCGGGTGGT3’

3304-3324

3304-3713

R-50 TGCCATGTAGACGACACTGCAGT3’

3691-3713

(410 bp)

F-50 CATCCACAACATTACTGGCG30

3868-3887

3868-4096

R-50 GTAATTAAAAAGCGTGCATGTG30

4075-4096

(228 bp)

F-50 CACCAAAAGAGAACTGCAATG30

86-105

86-508

488-508

(422 bp)

480-499 965-985

480-985 (505 bp)

E5 E6

0

R-5 TCGACCGGTCCACCGACCCCT3 E7

F-5’ATAATATAAGGGGTCGGTGG3’ R-5’CATTTTCGTTCTCGTCATCTG3’

81, 82 (MM4), 83 (MM7), 84 (MM8), IS39 and CP6108] (http://www.roche.com/products/product-details.htm?type= product&id=123). An additional primer pair targets the human b-globin gene (268-bp amplicon) as a control for cell adequacy, extraction and amplification [41]. DNA from 338 paraffin-embedded cervical biopsy samples was isolated and amplified with positive and negative controls, and HPV 16 was found in 124 cases, as a single infection. As part of the quality control for the Linear Array HPV Genotyping Test, 5 lL of the amplicon was assayed by electrophoresis in a 2 % agarose gel (Promega) with Tris boric acid EDTA (TBE) buffer (89 mM Tris boric acid, 2 mM disodium EDTA) (Promega) and visualized after ethidium bromide staining. If appropriate banding patterns were present, genotyping was performed [42] (Fig. 1). HPV genotyping results were interpreted according to the recommendation of the manufacturer after being visualized using the detectable hybridization bands (Fig. 2). Sequence analysis of HPV16 genes by PCR and direct sequencing PCR was performed using the cycling conditions described in Table 1, in a final volume of 50 ll using 50 pmol of each primer, and 2X PCR master mix (Fermentas, St. Leon-Rot, Germany). Different sets of primers were designed to cover the investigated HPV16 genes. The first cycle was preceded by DNA denaturation step for 5 minutes at 95 °C, followed by 34 cycles of denaturation, annealing and extension. The last cycle was followed by a 5 min elongation step at 72 °C. PCR products were analyzed by electrophoresis in a 2 % agarose gels. Purification was performed using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), and the DNA concentration was determined using a NanoDrop spectrophotometer.

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0

47 °C/60 s 50 °C/60 s 47 °C/45 s 47 °C/45 s 57 °C/60 s

DNA sequencing DNA sequencing was performed using 5-10 ng of purified DNA, 3.2 pmol of primer and an ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit. The forward and reverse primers were the same ones that were used for PCR reactions, generating PCR products with both orientations. The cycling conditions were as follows: 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 120 s at 60 °C. To remove unincorporated salts and BigDyeÒ Terminator (BDT), PCR products were purified using a BigDye XTerminator TM Purification Kit. HPV16 variants were determined by sequencing using an ABI PRISMÒ 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Alignment of HPV16 sequence variations and building of a phylogenetic tree The GenBank database [43] was queried to retrieve all available sequences for HPV16 subtypes and variants [44]; the retrieved sequences were saved in FASTA sequence format. The raw sequences were assembled using Velvet version 1.2.10 software. First, we compared each analyzed gene sequences and built a phylogenetic tree. The Clustal W2.0 algorithm, employing default parameters [45], was used for sequence alignment. To determine the percent sequence identity between the query and database sequences, Basic Local Alignment Search Tool [BLAST] pairwise alignments were used [46]. A maximum-likelihood phylogenetic tree (1000 bootstrap replicates) was constructed from the alignment of HPV16 sequences (built after target gene sequencing) derived from 68 CINs and 29 SCC samples and reference sequences. The GTR nucleotide substitution model was used for phylogenetic tree [47].

HPV16 variants associated with cervical neoplasia in Romania

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Statistical analysis Statistical analysis was performed using the v2 test (chi squared test) to determine the association between different E2, E4, E5, E6 and E7 HPV16 variants and the pathological status of the studied specimens. P-values less than 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Values are expressed as means ± SD. In order to determine the association between different E2, E4, E5, E6 and E7 HPV16 variants and the pathological status of the studied specimens, the v2 test was used. P-values less than 0.05 were considered statistically significant. Also, the Spearman correlation test was used to model the association between different gene variants, and statistical analysis was carried out using SPSS (version 13.0) software.

Results Study group characterization From the 338 paraffin-embedded cervical biopsy samples investigated, HPV DNA was detected in 231 cases (CINI, 23/104 cases; CINII, 71/89 cases; CINIII, 95/102 cases; SCC, 42/43 cases). In 124 cases, HPV 16 was present as a single infection: CINI, 21.7 % (5 cases); CINII, 40.8 % (29 cases); CINIII, 64.2 % (61 cases); SCC, 69 % (29 cases). The two geographical areas of CINs and SCC distribution are shown in Fig. 3. Coinfections (HPV16?HPV18? HPV31?HPV33) were detected in 74 of 231 cases, as follows: CIN I, 13/23 cases; CIN II, 34/71 cases; CIN III, 21/95 cases; SCC, 6/42 cases. Analysis of HPV16 E2 gene sequences HPV16 E2 variants were detected in 27 of 124 cases. In the transactivation domain, the substitutions C2859A (H35Q), G2890T (A46S), C3158A (T135K) and G3181A (A143T) were found, and within the hinge domain, A3361G (N203D), C3376G (P208A), T3386C (I211T), A3395G (H214R), C3409T (P219L), C3437A (A228D), G3448A (E232K), C3515A (T254N), T3516C (T254N), G3459A (Q235-), A3489G (P245-), A3537C (S261) and T3565G (F271V). In the DNA-binding domain, we found the substitutions C3683A (T310K), T3693A (T313-) and T3705C (S317-) (Table 2). Ninety-seven of the 124 isolates were of the prototype sublineage (EG-P), and 21 were of the AA sublineage, with both synonymous and non-synonymous nucleotide substitutions. The rest of the cases (6/124) were EG sublineage ‘‘prototype-like’’ with synonymous and

Fig. 3 Geographical areas of CINs and SCC distribution

non-synonymous nucleotide substitutions (but their presence did not change their sublineage assignment). Analysis of E4 HPV16 gene sequences E4 HPV16 variants were detected in 27 of 124 cases. Only silent mutations (positions 3361, 3386 and 3376) were detected in the N-terminal domain, while the C-terminal domain contained A3537C and T3565G missense mutations resulting in amino acid changes (Q69P and H78Q, respectively). Although mutations in the C-terminal domain are important for protein oligomerisation, the most important missense mutations were those located between the C-terminal and N-terminal domains: A3395G, C3437A, G3459A, A3489G, C3515A and T3516C. These substitutions result in amino acid changes (T22A, P36T, A43K, Q53R, L62I and L62P, respectively) and seem to be important in malignancy progression. [40] Two other silent nucleotide mutations at positions 3409 and 3448 (Table 2) were identified. Ninety-seven of 124 isolates were of the prototype sublineage (EG-P). Twentythree contained synonymous and non-synonymous nucleotides substitutions and were clustered as the AA sublineage. Four of the 124 cases were ‘prototype-like’’ (EG-P) with synonymous and non-synonymous nucleotides substitutions. The E4 coding region overlaps the E2 hinge region. Five nucleic acid variants lead to amino acid changes in both the E2 hinge domain and the E4 protein. (Table 2). Analysis of HPV16 E5 gene sequences The most prevalent missense mutations in HPV16 E5 found in this study were as follows: T3904A, T3910C, G3919C, T3928C, A3978C, T3988C, T3991G, T4033C, A4041G/C, leading to amino acid changes (F19I; V21A; C24S; L27P; I44L; L47S; L48A; V62A; I65V/L). Only one silent mutation (nt 4001) was detected (Table 3). Fifty-five

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Table 2 Analysis of HPV16 gene sequences in patients with cervical lesions of different grades

(a)

(b)

a)

E2 variants clustered in two sublineages: EG-P and AA

b)

E4 variants clustered in two sublineages: EG-P and AA

1)

Nucleotide variations in the E4 gene that resulted in nucleotide variations and amino acid substitutions in the E2 hinge region

2)

Nucleic acid variations in both E2-E4 HPV16 genes are shown in gray

n = Indicates the number of isolates identified for each sublineage * Mutations not leading to amino acid changes

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HPV16 variants associated with cervical neoplasia in Romania

3311

Table 3 Analysis of HPV16 oncogene sequences and sublineages in patients with cervical lesions of different grades

(a)

(b)

(c)

a)

E5 oncogene sequences with EG-P sublineage

b)

E6 oncogene sequences with As-P and EG sublineages

c)

E7 oncogene sequences with EG-P and AA sublineages

n = Indicates the number of isolates identified for each sublineage * Mutation not leading to amino acid changes

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Table 4 Frequencies of synonymous/non-synonymous changes for investigated genes correlated with malignancy grade HPV16 gene Grade of cervical lesions

E2 N/S

f

CIN I

0

0

CIN II

22/4

0.85/0.15

CIN III

27/4

0.87/0.13

SCC

55/9

0.86/0.14

1)

E2 hinge domain

E4

E5

E6

E7

N/S

f

N/S

f

N/S

f

N/S

f

N/S

f

0

0

0

0

8/0

1/0

11/1

0.92/0.08

1/2

0.33/0.67

18/3

0.86/0.14

8/12

0.4/0.6

11/0

1/0

36/3

0.92/0.08

3/3

0.5/0.5

18/3

0.86/0.14

8/12

0.4/0.6

14/1

0.93/0.07

24/3

0.89/0.11

3/5

0.38/0.62

39/6

0.87/0.13

21/25

0.46/0.54

16/1

0.94/0.06

24/2

0.92/0.08

6/7

0.46/0.54

N/S = Ratio between non-synonymous and synonymous changes; f = frequency

of the 124 isolates were of the prototype sublineage (EGP), and 69 were ‘‘prototype-like’’ (EG-P), with synonymous and non-synonymous nucleotides substitutions. Analysis of HPV16 E6 gene sequences The most prevalent missense mutations in HPV16 E6 (C279G, C285G, T288G, T295G, A296C, A305C, A306G, G328G, T350G) leading to amino acid changes (Q66P; A68G; V69G; D71E; K72Q; K75R; F76L; E82D; L90V) were found in 55 of 124 cases. One silent mutation was found at position 322 (I80I). E6 variants were clustered in two sublineages: 69 of 124 isolates were of the prototype sublineage (As-P), and 41 clustered as the EG sublineage. The other 14 were ‘‘prototype-like’’ (As-P), with synonymous and non-synonymous nucleotide substitutions. Two new missense mutations (C279G and A305C) associated with 350G or other E6 variants were identified in 14 of 55 and 36 of 55 cases, respectively (Table 3). Analysis of E7 HPV16 gene sequences The most prevalent missense mutations in HPV16 E7 (C749T and C790T) were found in 21 cases, leading to the amino acid changes S63F and R77C, respectively. Four silent mutations (positions 732, 789, 795, 822) were also detected. HPV16 E7 variants were clustered in two sublineages: 103 of the 124 isolates were of the prototype sublineage (EG-P), and 8 were included in the AA sublineage and displayed synonymous (732C, 795G) and nonsynonymous (749T) nucleotide substitutions. Thirteen of 21 cases were ‘‘prototype-like’’ (EG-P) with non-synonymous/synonymous substitutions (Table 3). Frequencies of synonymous/ non-synonymous changes In order to establish the effect of synonymous/non-synonymous mutations on malignant transformation, the relationship of the frequency of such mutations to lesion severity was investigated (Table 4). The frequencies of non-synonymous mutations for the E2, E5 and E6 HPV16 genes are much higher than for synonymous mutations, as

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compared with the E4, E7 HPV16 genes. We found a significant frequency of non-synonymous/synonymous mutations correlated with the severity of lesions (p [ 0.001). We observed that the frequency of non-synonymous mutations is higher for the E2, E5 and E6 genes, while for E4 gene, synonymous mutations are prevalent. Variants associated with CINs and SCC lesions All of the 124 HPV16 samples screened by the standard procedure were grouped according to histology and sequence variations. The degree of variation differed with histological grade, with a higher prevalence of variants being found in SCC. Although the SCC fraction for the E4 AA 3537C sublineage (0.5) was higher, only 3515A and 3516C (0.7) substitutions allow the EG-P and AA sublineages to be distinguished. The E7 AA 749T variant (0.5) distinguishes between the EG-P and AA sublineages. Although SCC fractions for E6 EG 285G (0.29), 296C (0.17), and 305C (0.24) substitutions were more frequently associated with As-P, only the EG 350G variant (0.36) permits the EG and As-P sublineages to be distinguished (Table 5). Correlation of variant patterns Statistical analysis revealed some correlations between the following genes: E6 and E5 (r = -0.390; p \ 0.0001); E7 and E4 (r = -0.331; p \ 0.0001); E7 and E5 (r = -0.276; p = 0.003). To establish if mutations in viral genes are associated with cervical cancer development, we compared the mutation patterns by grouping two different viral genes. In CINI-II cases, no correlation was observed when we compared the mutation patterns. In CINIII cases, a good correlation was observed for E5 and E6 (13/61), and E5 and E7 (8/61), (p \ 0.0001) when both genes were mutated, and a lower correlation was observed between E4/E2 hinge-E5 (5/56) and E4/E2 hinge-E6 (2/59) (p = 0.4392). In SCC cases, the best-correlated mutation patterns were obtained for E4/E2hinge-E5 (17/29 patients had mutations in both genes [r = 0.7984; p \ 0.0001]. A good correlation

HPV16 variants associated with cervical neoplasia in Romania

3313

Table 5 CINs and SCC lesions associated with HPV16 variants HPV16 gene

Sublineage

E2

EG-P

AA

E4

EG-P

HPV16 variant

CIN I f

CIN II CIN I fraction*

f

CIN III CIN II fraction*

f

SCC CIN III fraction*

f

SCC fraction*

2859

0

0

0

0

1

0.25

1

0.25

2890

0

0

0

0

1

0.25

0

0

3158 3181

0 0

0 0

1 0

0.25 0

1 2

0.25 0.5

1 1

0.25 0.25

3361

0

0

1

0.25

0

0

0

0

3376

0

0

1

0.25

1

0.25

1

0.25

3386

0

0

1

0.25

0

0

0

0

3395

0

0

0

0

0

0

0

0

3409

0

0

1

0.25

2

0.5

1

0.25

3437

0

0

0

0

0

0

0

0

3448

0

0

1

0.25

1

0.25

1

0.25

3459

0

0

0

0

0

0

0

0

3489

0

0

0

0

0

0

0

0

3515

0

0

0

0

0

0

0

0

3516

0

0

0

0

0

0

0

0

3537

0

0

1

0.25

0

0

0

0

3565

0

0

1

0.25

0

0

0

0

3683 3693

0 0

0 0

1 0

0.25 0

1 1

0.25 0.25

0 1

0 0.25

3705

0

0

1

0.25

0

0

0

0

2859

0

0

0

0

1

0.04

4

0.17

2890

0

0

0

0

3

0.13

3

0.13

3158

0

0

0

0

0

0

2

0.08

3181

0

0

1

0.04

0

0

8

0.34

3361

0

0

0

0

1

0.04

4

0.17

3376

0

0

1

0.04

4

0.17

11

0.47

3386

0

0

3

0.13

4

0.17

16

0.69

3395

0

0

0

0

0

0

2

0.08

3409

0

0

3

0.13

4

0.17

3437

0

0

0

0

0

0

3448

0

0

3

0.13

4

0.17

3459

0

0

0

0

0

3489

0

0

0

0

1

3515 3516

0 0

0 0

3 3

0.13 0.13

4 4

3537

0

0

3

0.13

3565

0

0

0

0

3683

0

0

3

0.13

3693

0

0

0

0

3705

0

0

0

0

3

0.13

1

0.04

3361

0

0

1

0.25

0

0

0

0

3376

0

0

1

0.25

1

0.25

1

0.25

3386

0

0

1

0.25

0

0

0

0

3395

0

0

0

0

0

0

0

0

3409

0

0

1

0.25

2

0.5

1

0.25

3437

0

0

0

0

0

0

0

0

12

0.52

2

0.08

12

0.52

0

2

0.08

0.04

2

0.08

0.17 0.17

16 16

0.69 0.69

4

0.17

12

0.52

3

0.13

1

0.04

1

0.04

15

0.65

0

0

2

0.08

123

3314

A. Plesa et al.

Table 5 continued HPV16 gene

Sublineage

AA

E5

E6

EG-P

As-P

EG

123

HPV16 variant

CIN I

CIN II

f

CIN I fraction*

3448

0

0

1

0.25

1

0.25

1

0.25

3459

0

0

0

0

0

0

0

0

3489 3515

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

3516

0

0

0

0

0

0

0

0

3537

0

0

1

0.25

0

0

0

0

3565

0

0

1

0.25

0

0

0

0

3361

0

0

0

0

1

0.04

4

0.17

3376

0

0

1

0.04

4

0.17

11

0.47

3386

0

0

3

0.13

4

0.17

16

0.69

3395

0

0

0

0

0

0

2

0.08

3409

0

0

3

0.13

4

0.17

3437

0

0

0

0

0

0

3448

0

0

3

0.13

4

0.17

12

0.52

3459

0

0

0

0

0

0

2

0.08

3489

0

0

0

0

1

0.04

2

0.08

3515

0

0

3

0.13

4

0.17

16

0.69

3516 3537

0 0

0 0

3 3

0.13 0.13

4 4

0.17 0.17

16 12

0.69 0.52

f

CIN III CIN II fraction*

f

SCC CIN III fraction*

f

SCC fraction*

12

0.52

2

0.08

3565

0

0

0

0

3

0.13

1

0.04

3904

1

0.01

0

0

0

0

1

0.01

3910

1

0.01

1

0.01

1

0.01

1

0.01

3919

1

0.01

1

0.01

1

0.01

1

0.01

3928

0

0

0

0

1

0.01

1

0.01

3978

3

0.04

8

0.11

28

0.4

13

0.18

3988

0

0

0

0

1

0.01

2

0.03

3991

1

0.01

4

0.05

4

0.05

1

0.01

4001

0

0

0

0

1

0.01

1

0.01

4033

0

0

2

0.03

1

0.01

3

0.04

4041

3

0.04

13

0.18

31

0.44

16

0.23

279

1

0.07

3

0.21

3

0.21

2

0.14

285

0

0

4

0.28

5

0.35

4

0.28

288

0

0

1

0.07

0

0

0

0

295 296

0 1

0 0.07

2 2

0.14 0.14

3 2

0.21 0.14

2 2

0.14 0.14

305

1

0.07

2

0.14

2

0.14

2

0.14

306

0

0

1

0.07

1

0.07

1

0.07

322

0

0

2

0.14

2

0.14

1

0.07

328

0

0

1

0.07

0

0

0

0

350

0

0

0

0

0

0

0

0

279

1

0.02

2

0.04

1

0.02

2

0.04

285

3

0.07

12

0.29

6

0.14

12

0.29

288

1

0.02

8

0.19

1

0.02

2

0.04

295

0

0

0

0

0

0

0

296

1

0.02

11

0

0.26

2

0.04

7

0.17

305

1

0.02

13

0.31

5

0.12

10

0.24

HPV16 variants associated with cervical neoplasia in Romania

3315

Table 5 continued HPV16 gene

E7

Sublineage

EG-P

AA

HPV16 variant

CIN I

CIN II

f

CIN I fraction*

306

0

0

322

1

0.02

328 350

0 3

0 0.07

732

0

0

749

0

789

0

790

f

CIN III

SCC

CIN II fraction*

f

CIN III fraction*

f

SCC fraction*

0

0

0

0

0

2

0.04

1

0.02

2

0.04

0 14

0 0.34

0 9

0 0.21

0 15

0 0.36

0

0

0

0

1

0.07

0

2

0.15

2

0.15

5

0.38

0

1

0.07

2

0.15

3

0.23

0

0

1

0.07

3

0.23

2

0.15

795

0

0

1

0.07

0

0

1

0.07

822

0

0

0

0

5

0.38

4

0.30

732

1

0.12

0

0

3

0.37

4

0.5

0

749

1

0.12

0

0

3

0.37

4

0.5

789

0

0

0

0

0

0

0

0

790

0

0

0

0

0

0

0

0

795

1

0.12

0

0

3

0.37

4

0.5

822

0

0

0

0

0

0

0

0

*CINI-III, SCC/total case number for each; f = frequency

was obtained for E6 and E7 genes, with 9 out of 29 patients presenting the same mutation patterns (r = 0.5263; p = 0.0034). Less correlation was observed between E5 and E7. When we compared the mutation patterns by grouping three different viral genes, the most frequently mutated genes in SCC samples were E4/E2 hinge, E5, and E6 (p = 0.0004) (Fig. 4). None of the samples contained mutations in all of the sequenced genes (Fig. 4). Phylogenetic tree of CINs and SCC HPV16 variants Taking into account the prevalence of variants in CIN (67) and SCC (29) cases, we compiled two phylogenetic trees for establishing the main lineages with essential roles in malignant transformation. After target gene sequences were assembled, we observed that CINs were derived from EG and formed two clusters: the first (37/67 cases) was from Moldavia (28 EG and 9 AA), and the second (30/67 cases) was from Bucharest (26 EG and 4 As) (Fig. 5a). Similar results were found for SCC cases related to EG strains. The cluster corresponding to patients from the Bucharest area included 11of 29 cases, and the second, corresponding to patients from the Moldavia area, included the rest of the cases (18/29) (Fig. 5b).

Discussion Among HPVs, HPV16 is most frequently associated with cervical cancer, and its genome was one of the first sequenced. HPV16 variants have different oncogenic potential, and this may be due to changes in the biological properties of the virus and its transforming potential, increasing the risk of development of cervical cancer. Infection with certain HPV16 variants may lead to faster development of the disease [48, 49]. The increased oncogenicity of HPV16 variants is manifested before integration of the HPV genome into the host cell, most probably by reduced recognition by the host immune system. HPV variants have been identified by comparing the sequences of E2, E4, E5, E6 and E7 genes from our samples with prototype ones. HPV16 variants differ in prevalence and biochemical and biological properties, with uncertain implications in cervical cancer etiology. Eriksson et al. [48] did not report amino acid mutations in the E4 gene, but Roberts et al. [50], McIntosh et al. [51] and Tsakogiannis et al. [40] highlighted the role of some point mutations in E4 on the oncogenic potential of HPV16. Our results confirm previous preliminary studies suggesting that amino acid changes (T22A, P36T, A43K, Q53R, L62I and L62P) resulting from E4 nucleotide sequence variations (A3395G, C3437A, G3459A, A3489G, C3515A and T3516C) seem to be important in malignancy

123

3316

A. Plesa et al.

Fig. 4 Mutation patterns. a) Association of mutation patterns by grouping two viral genes in CINIII (p\0.0001; v2 = 27.6); b) association of mutation patterns by grouping two viral genes in SCC (p\0.0001; v2 = 36,63); c) association of mutations patterns by grouping three viral genes in CINIII; d) association of mutation patterns by grouping three viral genes in SCC (p = 0.0005; v2 = 17.58)

progression. We observed that some of the nucleotide variations observed in the E4 gene resulted in nucleotide variations and amino acid substitutions in the E2 hinge region. We found a significant association between the 3683A (T310K) variant in the E2 DNA-binding domain and high-grade histology (15 of 29 SCC cases had this mutation) [52]; the 350G E6 variant segregated with the E2 hinge region 3409T variant (7/29 cases) and with the E2 DNA-binding domain 3683A variant (6/29 cases), suggesting that infections with HPV16 containing those variants may be important in the progression toward highgrade intraepithelial disease. We also found that the E5 mutations T3904A, T3910C, G3919C, T3928C, A3978C, T3988C, T3991G, T4033C, A4041G/C are often present. Several combinations of these mutations were found more than once. Most E5 missense mutations (20/29) were found in cervical cancer. The large number of E5 sequence variations detected in precursor lesions (49/95) suggested that E5 plays an important role in the initial stage of cervical cancer. The fact that HPV DNA is usually identified in integrated forms and E5 is generally not expressed in cervical cancers has been taken as evidence that E5 is not essential for cervical cancer [53–55]. However, E5 variations in particular positions (3978C, 4041G) may be subsequently spread in a population [56]. These variations could be involved in persistence of HPV infection and in progression toward cancer and thus play a role in malignancy. Due to the low mutation rate of HPV, the presence of E4/E5 mutations might be a consequence of natural variation, rather than arising in a cancer genome, but HPV16 seems to evolve faster than expected from its genome size [57].

123

Sequence analysis has shown that a single nucleotide substitution in the HPV16 E6 gene could affect malignant transformation. Some studies have suggested that the 350G and 350T variants vary in their distribution between cervical cancers and controls [18, 58]. The 350G variant (the most common variant found in cervical cancer patients) is associated with an increased risk of persistent infection and with histological progression toward CIN II-III. This variant contains a change from leucine to valine at amino acid position 90, located in a potential epitope region [59]. These results suggest that viral variants might contribute to carcinogenesis or evasion of the host immune system. An interesting observation in our study was the higher prevalence of synonymous mutations in the E4 and E7 genes. Currently, there is an increasing body of evidence showing that synonymous mutations are not always silent and may impact various disease phenotypes [60]. A synonymous mutation in the E4 gene could nevertheless result in an amino acid change in the E2 protein. Changes occurring in the E2 hinge region have been observed to occur more frequently than in the conserved N- and C-terminal regions. When we compared mutation patterns in SCC samples, 17 of 29 cases had both E2 and E5 gene mutations, while 10 of 29 patients had the same mutation pattern for both E6 and E7 genes. None of the samples had mutations in all of the investigated genes, but the most strongly correlated mutation patterns were for the E2, E5 and E6 genes. Not all variations in the E2, E4, E5, E6 and E7 sequences are required for classification in one of the three sublineages described. For example, we observed that a 749T variant was misclassified as belonging to the AA

HPV16 variants associated with cervical neoplasia in Romania

3317

Fig. 5 Phylogenetic trees. Viral variants were identified by comparing EG-P sequences. All sublineages derive from the EG-P (AF536179:0.00483) and are segregated in two branches: one from

Bucharest and the other from Moldavia. a) CINs HPV16 variants; b) SCC HPV16 variants. Sample identification numbers are shown at the right of each branch; numbers above branches represent score values

sublineage based on the E7 sequence only but clearly belonged to the EG sublineage when E6 350G variation was taken into account.

After all target genes sequences (from CIN and SCC samples) were assembled, all patients were found to be infected with HPV16-EG, and two clusters were identified.

123

3318

The first (55/96 variants) was from Moldavia, and the second (41/96 variants) was from Bucharest. Nucleotide sequence analysis of HPV16 genes (from CINs) revealed that, from the Bucharest area, 26 of 30 EG cases were associated with the non-synonymous nucleotide substitution T ? G (arginine ? valine) at position 350 of E6, and 4 of 30 cases were of the As sublineage. The T ? G 350 nucleotide substitution in E6 gene allows the EG and As-P sublineages to be distinguished. In the Moldavia area, previous classifications based on E6 alone had identified isolates as belonging to the As lineage. Twenty-eight of 37 samples were related to EG-P with a non-synonymous nucleotide substitution T ? G, (arginine ? valine) (in position 350 of the E6 gene), while 9 of 37 samples were related to AA with the non-synonymous nucleotide substitutions C ? A (leucine ? isoleucine) and T ? C (leucine ? proline) (at positions 3515 and 3516 of the E4 gene, respectively). The nucleotide substitution T ? G 350 in the E6 gene allows the EG and AA sublineages in the Moldavia area to be distinguished. We were particularly interested in HPV16 variants infecting women with SCC. Nucleotide sequence alignment of E2, E4, E5, E6 and E7 sequences revealed that all sequences corresponded to the EG sublineage. In CINs and SCC, along with known point mutations in the identified variants, two new missense mutations in the E6 gene (C279G and A305C) were detected (together or alone) in 44 of 124 cases. These mutations were distributed as follows: CIN I, 4/5 cases (all in the Bucharest area); CIN II, 18/29 cases (14 in the Bucharest area); CIN III, 10/61 cases (7 in the Bucharest area); and SCC, 14/29 cases (9 in Bucharest). Nucleotide sequence analysis of the E4 gene and the ability of E6 to distinguish these sublineages are important for epidemiological studies, since it is particularly the AA sublineage that has been suggested to be associated with high-grade cervical intraepithelial neoplasia. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area (31.49/100,000 according to the Regional Center for Public Health in Iassy). For future larger-scale epidemiological studies, and because it is important to have a complete and standardized classification of HPV16 variants worldwide, we hope that our results highlight the important role of HPV16 variants during cervical carcinogenesis in Romania. In order to demonstrate the importance of these variants, more studies are needed, but to identify more-aggressive variants in HPV16-positive patients, newer tests for diagnosis are necessary. The new era of next-generation sequencing could bring new data regarding this aspect and could also be a tool for diagnosis in such patients. Acknowledgements This study was supported by the 41-081 and 41-030 Romanian Grants.

123

A. Plesa et al. Conflict of interest

None to declare.

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