E7 variants in cervical neoplasia in Chinese women

Gynecologic Oncology 119 (2010) 436–443

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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o

Distribution of human papillomavirus 58 and 52 E6/E7 variants in cervical neoplasia in Chinese women Tian Ding a, Xinyu Wang a, Feng Ye a, Xiaodong Cheng a, Ding Ma b, Weiguo Lu a, Xing Xie a,⁎ a

Women's Reproductive Health Laboratory of Zhejiang Province and Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, People's Republic of China Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, People's Republic of China

b

a r t i c l e

i n f o

Article history: Received 2 July 2010 Available online 25 September 2010 Keywords: Human papillomavirus (HPV) Cervical intraepithelial neoplasia (CIN) HPV variants HPV58 HPV52

a b s t r a c t Objectives. The specific intratype HPV genome variations may be associated with the development of cervical cancer in specific geographic regions and a given population. Human papillomavirus (HPV) 58 and 52 have been found to be relatively prevalent among Asian women including Chinese women. This study aimed to assess the risk of HPV 58 and 52 variants for cervical cancer and its precursors in Chinese women. Methods. A total of 2021cervical samples were collected. After DNA extraction and genotyping, a total of 298 (177 HPV58-single positive and 121 HPV52-single positive) DNA samples were analyzed for E6 and E7 sequence variations by direct sequencing. Results. A total of 29 new reported variations of HPV 58 and 52 were found. For HPV58, the presence of C632T (T20I) and G760A (G63S) variants in E7 showed a positive trend of the association with the severity of neoplasia (Ptrend b 0.05, χ2 test for trend). Conclusions. These findings suggest that C632T (T20I) and G760A (G63S) variants in HPV58 E7 are probably risk factors associated with the development of cervical cancer in Chinese women. The presence of HPV58/52 E6 and E7 variants may be different in Chinese women. © 2010 Elsevier Inc. All rights reserved.

Introduction It has been established by molecular biological and epidemiological studies that infection with specific types of genital human papillomavirus (HPV) is an essential etiological factor for both cervical cancer and its precursor lesions, cervical intraepithelial neoplasia (CIN) [1–3]. HPV16 and 18 are the most common cancer-associated types, accounting for about 70% of all cervical cancers worldwide whereas HPV 58 and 52 infections only for 0–3% [4]. However, accumulated studies have shown that HPV 52 and 58 are relatively more frequent among HPV-positive Chinese and other Asian women and there is 11.5–28% of prevalence across the full spectrum of cervical neoplasia in Chinese and Asian women [5–9]. It has been estimated that worldwide HPV prevalence is 10.4% in women with normal cervical cytology [10], but less than 1% of the women develop malignant lesions [11]. It is clear that persistent infection with specific high-risk HPV types is a strong marker for progressive CIN disease [12], whereas recent studies have revealed that persistence of highrisk HPV infection might be associated with virus intratype variants. The HPV intratype variants are defined as having nucleotide sequence variations no more than 2% in the coding region and 5% in

⁎ Corresponding author. Fax: + 86 57187061878. E-mail address: [email protected] (X. Xie). 0090-8258/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2010.08.032

the non-coding regions of the viral genome with respect to the prototype [13]. Concerning HPV intratype variants, the most extensive studies have been conducted on HPV16, followed by HPV18 [14]. It has been found that HPV16 included five major phylogenetic branches: European (E), Asian (As), Asian-American (AA), African-1 (Af1), and African-2 (Af2), and a few minor branches such as North American-1(NA1) [15]. These variants may differ among certain biologic and biochemical properties. The evidence that specific intratype HPV genome variations may influence persistence of viral infection and cervical lesions progress from precursor lesion to cancer was revealed by independent studies [16–18]. The study by Londesborough and his colleagues indicated that persistent HPV16 infection and/or cervical high-grade lesions were predominantly associated with the variant T350G (L83V, leucine → valine) where the Arabic numerals represented the nucleotide or amino acid position and the letter preceding this number refers to the reference base or amino acid derived from the prototype sequence and the letter following it refers to its substitution. HPV onco-proteins E6 and E7 are essential factors for HPV-induced cellular immortalization, transformation, and carcinogenesis. The studies from European and American women have shown that specific HPV16 E6 and E7 variants may carry a higher risk for the development of invasive cervical cancer and cervical intraepithelial neoplasia in a given population [15,16,19,20]. The current data on HPV variants are derived mainly from HPV16, the most common type worldwide,

T. Ding et al. / Gynecologic Oncology 119 (2010) 436–443

whereas there are few reports on HPV 58 or 52 variations. Considering relatively higher prevalence of HPV 58 and 52 in Chinese and Asian women, we investigated the sequence variations of HPV58 and 52 in Chinese women and analyzed the association between viral intratype variations and cervical cancer, as well as CIN, so as to explore influence of HPV 58 and 52 variations on the development of cervical cancer.

Materials and methods Study subjects and sample collection During April 2008 and December 2009, a total of 2021 HPV positive cervical samples were collected from colposcopy clinic in Women's Hospital, Medical School, Zhejiang University, eastern China. The population studied ranged in age of 21–73 years with a mean of 38 years. The study was approved by the Women's Hospital Ethical Committee and informed consent was taken from each of the subjects. All subjects were examined by colposcopy and biopsy samples were taken from suspicious lesions for pathological diagnosis and DNA extraction. Cervical disease status was defined on the basis of biopsy findings. Case biopsies were graded as CIN1, CIN2–3, or invasive cervical carcinoma (ICC) according to the highest grade present within a lesion. The cervical exfoliated cell samples alone were obtained from women with normal colposcopy results and were classified as the controls. The biopsy tissues confirmed as normal squamous epithelial or no any degrees of CIN by pathologic diagnosis were also classified as controls. Women who either were pregnant or had undergone cervical conization were excluded from the study. The collected fresh biopsy specimens were rinsed in cold saline water and immediately stored in liquid nitrogen at least 24 h preventing nucleotide degradation, then stored at −70 °C in a deep freezer for further processing later. Cytological samples were centrifuged and then stored at −70 °C for DNA sequencing. A cervical-exfoliated cell sample was collected with DNAPap cervical sampler (Digene) from each subject stored at 4 °C for HPV detection and typing.

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PCR amplifications and cycle sequencing The obtained DNA extracted from HPV58 and 52 positive samples was amplified for E6 and E7 respectively with primers listed in Table S1. A total of 50 μl polymerase chain reaction (PCR) reaction mix containing 5 μl template DNA, 10× PCR buffer, 2.5 mM dNTPs, 25 mM MgCl2 (TaKaRa Biotechnology Co., Ltd.), 10 μmol of each primer (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.), and 0.3 U of Taq polymerase (TaKaRa Biotechnology Co., Ltd.) run for 40 cycles in the GeneAmp PCR System PTC-200 (Bio-Rad) with 94 °C denaturation (45 s), 55 °C or 64 °C anneal (45 s) and 72 °C extension (45 s), including initial denaturation of 5 min and final extension of 7 min resulting products for E6 and E7, respectively. The amplicon was purified with absolute ethanol-EDTA (125 mM, pH 8.0)–NaAC (pH 5.2–5.4)–70% ethanol method. The PCR product was purified twice, then sequenced with Bigdye Terminator v3.1Cycle Sequencing Kit (Applied Biosystems), according to the instruction and the same primer as the above. The mix was run for 40 cycles in the PTC-200 (Bio-Rad) with 96 °C denaturation (10 s), 50 °C anneal (5 s) and 60 °C extension (4 min), including initial 96 °C denaturation of 1 min. The fluorescent dye dideoxy termination product was performed on automated sequencer (ABI 3100 Prism, Applied Biosystems). In approximately half of the samples were repeated PCR and sequencing from both directions to exclude PCR artifacts. Variants identification and statistical analysis The sequenced product was aligned with prototype sequence on NCBI (http://blast.ncbi.nlm.nih.gov/Blast). The HPV 58 prototype (GenBank Accession No. D90400) and the HPV52 prototype (GenBank Accession No. NC_001592) were used as the standard for comparisons and nucleotide position numbering respectively [29]. For examining distributions of HPV 58/52 variations with respect to disease severity, the Linear-by-Linear Association (Ptrend) and Fisher's exact test (P) or Pearson χ2 (P) were employed [29]. Two-sided, P b 0.05 was considered to be statistically significant. Results Characteristics of the study subjects and HPV58/52 distribution

DNA extraction and HPV typing The genomic DNA of frozen cervical biopsy specimens and scrape cells was extracted by the standard method of proteinase K digestion and phenol–chloroform abstraction. The lapped biopsy tissue with tissue grinder and cervical scrape cells were digested with 1 ml of lysis buffer (Tris–HCl 10 mM pH 8.0, EDTA 0.1 M pH 8.0, SDS 0.5%, Proteinase K 100 μg/ml ) at 55 °C for1–3 h, then, extracted with phenol–chloroform precipitations as described by Lizano et al. [21]. The obtained DNA was amplified for a segment of 268 bp of human β-globin (GH20/PC04) (Table S1) gene to check the quality of the DNA extracted [22]. Only the DNA amplified by β-globin was prepared for the HPV typing. Before typing, the HC II assay was performed according to manufacturers' instructions with specific HR-HPV RNA probe “cocktails” simultaneously detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 to detect HPV positive specimens. Samples were considered positive when the ratio of relative light units compared to standard positive control was equal or greater than 1.0 [23]. For the HPV genotyping, flow-through hybridization (HPV GenoArray test kit; Hybribio Ltd., Hong Kong) [24] was used based on the principle of Reverse Dot Blot Assay which was described in previous studies [25]. The HPV typing method could classify 21 HPV subtypes including 12 established high-risk (HR) types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59), 3 probably HR types (HPV-53, -66, -68), 6 established low-risk (LR) types (HPV-6, -11, -42, -43, -44, and CP8304) [26–28].

A total of 2021 samples from Chinese women with HR-HPV positive were enrolled in the study, including 1597 women with tissue samples and 424 women with cytology samples. Of those, there were 177 samples with single HPV58 positive and 121 with single HPV52 positive after HPV genotyping. Samples with multiple HPV infections were excluded to avoid confounding the results. In all single HPV58 and 52 positive subjects, 187 (62.8%), median age 37 years (range 22–57), were normal controls including 92 HPV58 positive and 95 HPV52 positive samples; 49 (16.4%), median age 36 years (range 24–48), were cervical intraepithelial neoplasia (CIN) grade I including 36 HPV58 positive and 13 HPV52 positive samples; 57 (19.1%), median age 39 years (range 25–51), were CIN 2–3 including 45 HPV58 positive and 10 HPV52 positive samples; and 5 (1.7%), median age 44 years (range 24–73) were ICC including 4 HPV58 positive and 1 HPV52 positive samples (Tables 1 and 2). Distribution of HPV58 E6 and E7 variants in different cervical samples In normal and different cervical lesions, a total of 10 variations of E6 and 21 variations of E7 at the nucleotide lever were identified compared with the prototype (No. D90400), shown in Tables 1 and 2. Not all genes could be amplified or sequenced and it may be because the copy of HPV infection was low or the amplicon was unstable. Five of 10 E6 variants and 14 of 21 E7 variants were non-synonymous mutations leading to amino acid variations that were considered to

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Table 1 HPV58E6 sequence variations in different cervical samples. Variants distribution

2

No. (%) of women with cervical neoplasia

Nucleotide sequence variation

Amino acid substitution

− T114C C187T G203C A259G C307T C317T C367A A388C G432A A479C Total

− F2S I26I E32Q L50L C66C L70L D86E K93N R108K R124R

Normal

CIN1

CIN2–3

ICC

Total

84

35

44

4

167

14(16.7) 0 0 1(1/84) 2(2.4) 32(38.1) 1(1/84) 1(1/84) 37(44.0) 0 0 88

10(28.6) 1(1/35) 0 1(1/35) 1(1/35) 10(28.6) 0 1(1/35) 14(40) 0 1(1/35) 39

9(20.5) 0 1(1/44) 0 2(4.5) 16(36.4) 0 0 14(31.8) 0 0 42

0 0 0 0 0 1(25) 0 0 3(75) 1(25) 0 5

3(19.8) 1(1/167) 1(1/167) 2(1.2) 5(3.0) 59(35.3) 1(1/167) 2(1.2) 68(40.7) 1(1/167) 1(1/167) 174

P

Ptrend

0.338 (–) (–) (–) 0.794 0.756 (–) (–) 0.286 (–) (–)

0.48 (–) (–) (–) 0.514 0.632 (–) (–) 0.493 (–) (–)

Note: CIN, cervical intraepithelial neoplasia; ICC, invasive cervical cancer. Ptrend, by χ linear-by-linear association; P, Pearson or Fisher's exact test; −, no changes compared with the prototype; a dash (–) indicates that there were too few isolates for statistical analysis.

have functional difference from the prototype. A388C was the most frequent non-synonymous mutation, leading to the change of amino acid K93N. C307T was the most frequent synonymous mutations of E6. In E7 variants, G760A and G761A led to the same genetic code change but expressed different amino acids G63S and G63D which occurred in 18.8% and 15% included samples respectively. T803C was the most common non-synonymous mutation, leading to the change of amino acid V77A. T744G was the most common synonymous mutation. The prototype of E6 and E7 was identified in 19.8% and 28.1% of the respective total sequenced samples. The presence of C632T (T20I) and G760A (G63S) variants in E7 showed a positive trend of the association with the severity of neoplasia (Ptrend b 0.05, χ2 test for trend). The other variations in E6 and E7, shown in Table 1, were too few for statistical analysis. The total HPV58 positive samples were grouped into 23 different variants and named according to their frequency of occurrence as ZJ1 to ZJ23, respectively (Fig. 1).

Distribution of HPV52 E6 and E7 variants in different cervical samples At the nucleotide lever, 11 variations in HPV52 E6 and E7, respectively, were identified. Of these variations, 5 in E6 and 7 in E7 were non-synonymous. The amino acid substitutions were shown in Tables 3 and 4. The most frequent variation in E6 was the 93rd genetic code, which led to two amino acid changes: K93Q and K93R. The corresponding nucleotide changes were A378C, A378G and A379G. C751T and A801G were the two most frequent synonymous variations in E7, but no significant trends accordant with severity of cervical neoplasia were observed. According to the BLAST results, there was a guanine insertion between positions 705 and 706, while a deletion of guanine between 706 and 707. Before the position 705 and after 707, there were no similar mutations. So, it only led to the 52nd codon of E7 change from GAC to AGC so the encoded amino acid changes from serine to aspartic acid. This mutation was found in two HPV 52

Table 2 HPV58E7 sequence variations in different cervical samples. Variants distribution

No. (%) of women with cervical neoplasia

Nucleotide sequence variation

Amino acid substitution

− C595T G599A G613A C632T G694A T726C T744G C753T G760A G761A A793G C798T C801A T803C G806C G819A T824A T845G C850G G854A G954A Total

− L8L R9K D14N T20I G41R A51A T57T Y60Y G63S G63D T74A T75T D76E V77A R78P Q82Q L84H V91G P93A S94N Q97Q

Normal

CIN1

CIN2–3

ICC

Total

82

34

41

3

160

22(26.8) 0 5(6.1) 0 11(13.4) 14(17.1) 0 56(68.3) 1(1/82) 10(12.2) 15(18.3) 1(1/82) 1(1/82) 1(1/82) 28(34.1) 0 0 1(1/82) 0 0 0 0 166

10(29.4) 1(1/34) 2(5.9) 1(1/34) 6(17.6) 4(11.8) 0 18(52.9) 0 7(20.6) 4(11.8) 1(1/34) 1(1/34) 1(1/34) 9(26.5) 1(1/34) 1(1/34) 0 0 1(1/34) 0 1(1/34) 69

11(26.8) 0 0 0 13(31.7) 4(9.8) 2(4.9) 27(65.9) 0 13(31.7) 5(12.2) 0 0 0 8(19.5) 0 0 0 1(1/41) 0 1(1/41) 0 85

2(66.7) 0 0 0 0 0 0 1(33.3) 0 0 0 0 0 0 1(33.3) 0 0 0 0 0 0 0 4

45(28.1) 1(1/160) 7(4.4) 1(1/160) 30(18.8) 22(13.8) 2(1.3) 102(63.8) 1(1/160) 30(18.8) 24(15) 2(1.3) 2(1.3) 2(1.3) 46(28.8) 1(1/160) 1(1/160) 1(1/160) 1(1/160) 1(1/160) 1(1/160) 1(1/160) 324

P

Ptrend

0.532 (–) (–) (–) 0.05 0.497 (–) 0.292 (–) 0.033 0.549 (–) (–) (–) 0.346 (–) (–) (–) (–) (–) (–) (–)

0.624 (–) (–) (–) 0.019 0.249 (–) 0.389 (–) 0.01 0.329 (–) (–) (–) 0.143 (–) (–) (–) (–) (–) (–) (–)

Note: CIN, cervical intraepithelial neoplasia; ICC, invasive cervical cancer. P2trend, by χ linear-by-linear association; P, Pearson or Fisher's exact test; −, no changes compared with the prototype; a dash (–) indicates that there were too few isolates for statistical analysis.

T. Ding et al. / Gynecologic Oncology 119 (2010) 436–443 Fig. 1. Nucleotide sequence variations in the E6 and E7 open reading frames (ORFs) detected in135 human papillomavirus (HPV) 58 positive Chinese women. HPV58 prototype (GenBank accession number No. D90400) was used as the reference. Nucleotide positions where variations were detected are written across the top. Dashes indicated positions at which no variation was found. Lowercase letters: a silent nucleotide variation; Uppercase letters: a missense variation. The position of amino acid change is stated numerically following it refers to its substitution. GenBank accession numbers for sequences reported in this paper are HM004104–HM004108 for E6 sequences and HM004109–HM004114 for E7 sequences. 439

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Table 3 HPV52E6 sequence variations in different cervical samples. Variants distribution

No. (%) of women with cervical neoplasia

Nucleotide sequence variation

Amino acid substitution

T107C T144C G168A T191C G350T G356A A378C A378G A379G C467A A530G Variations total

F2F L15L V23M C30C A83A G85G K93R K93G K93R N122K R143R

Normal

CIN1

CIN2–3

ICC

Total

91

13

12

1

117

1(1/91) 1(1/91) 1(1/91) 0 91(100) 18(19.78) 2(2.20) 1(1/91) 91(100) 1(1/91) 1(1/91) 208

0 0 0 0 13(100) 1(1/13) 1(7.69) 0 13(100) 0 0 28

0 0 0 2(16.67) 12(100) 3(25) 0 0 12(100) 0 1(1/12) 30

0 0 0 0 1(100) 0 0 0 1(100) 0 0 2

1(1/117) 1(1/117) 1(1/117) 2(1.7) 117(100) 22(18.8) 3(2.6) 1(1/117) 117(100) 1(1/117) 2(1.7) 268

P

Ptrend

(–) (–) (–) (–) (–) 0.497 0.521 (–) (–) (–) (–) (–)

(–) (–) (–) (–) (–) 0.995 1.00 (–) (–) (–) (–) (–)

Note: CIN, cervical intraepithelial neoplasia; ICC, invasive cervical cancer. P2trend, by χ linear-by-linear association; P, Pearson or Fisher's exact test; −, no changes compared with the prototype; a dash (–) indicates that there were too few isolates for statistical analysis.

positive samples, one was normal control and the other was CIN1. The total HPV52 positive samples were grouped into eight different variants and named according to their frequency of occurrence as ZJHPV52-1 to ZJHPV52-8, respectively (Fig. 2). Discussion It has been revealed that the specific E6 and E7 variants in HPV16 and 18, most common types accounting for about 70% of all cervical cancers worldwide, may carry a higher risk for the development of invasive cervical cancer and its precursors in a given population [15,20,30,31]. HPV58 and 52 are relatively prevalent genotypes in Asian including Chinese women [8]. Recently, a few studies have analyzed the sequence variations in E6 and E7 genes of HPV 58 and 52, and found their risk association with the development of cervical neoplasia [29,32–34]. In this study, we found that a total of 21 sequence variations in E7 and 10 in E6 for HPV58, E7 variations occurred higher than E6 variations. Moreover, we firstly detected 10 variants in E7 and 5 in E6 of HPV 58, which were not reported before to our knowledge. Those newfound variations included T114C(F2S), A259G, C317T, G432A (R108K), and A479C in E6; C595T, G613A(D14N), C753T, G806C (R78P), G819A, T824A (L84H), T845G (V90G), C850G (P93A), G854A (S94N), and G954A in E7. The frequencies of other variations except

for A259G were very low and mainly occurred in normal women or CIN1 patients. A388C (K93N) was the most frequent variant in HPV58 E6 in our population. It was previously reported that frequency of A388C (K93N) presented a statistically significant negative trend accordant with severity of cervical neoplasia and ICC in HPV58 positive samples from Hong Kong, China. We found that the variant A388C (K93N) was present in 75% ICC in this study but there were no statistically significant differences among CIN1 and 2–3 (P = 0.450). Our result was in accordance with that reported by Wu et al. They found that A388C (K93N) was the most common variant present in 45.9% cervical cancer specimens. The three most frequent variations of HPV58 E7 were T744G and T803C (V77A), as well as the prototype, accounting for 63.8%, 28.8%, and 28.1% respectively, in whole sequenced cervical samples but no statistical significances were found, just as previous reports. In the report by Chan et al. [34], T744G and T803C (V77A) were found in 19 and 17 of 37 cervical cancer specimens, respectively, but not significantly different among cervical samples. Additionally, we found that E7 C632T (T20I) and G760A (G63S) variants were apparently linked except for one isolate from CIN1 subject that had the G760A (G63S) substitution in the absence of C632T (T20I). It has been shown that E7 C632T (T20I) and G760A (G63S) variants were apparently linked. For instance, Chan et al. reported that only four subjects with normal cervix had the G760A (G63S) substitution

Table 4 HPV52E7 sequence variations in different cervical samples. Variants distribution

No. (%) of women with cervical neoplasia

Nucleotide sequence variation

Amino acid substitution

T573A G592T A599G C662T T727G C733T G742A C751T A801G T848G A849C Variations total

T7T D14Y Q16R T37I Y59D H61Y D64N L67L Q83Q L99R L99L

Normal

CIN1

CIN2–3

CA

Total

81

13

12

0

106

1(1/81) 1(1/81) 1(1/81) 1(1/81) 1(1/81) 1(1/81) 1(1/81) 81(100) 81(100) 1(1/81) 0 170

0 0 0 0 0 0 1(1/13) 13(100) 13(100) 0 1(1/13) 28

0 0 0 1(1/12) 1(1/12) 1(1/12) 1(1/12) 11(91.67) 12(100) 1(1/12) 0 28

0 0 0 0 0 0 0 0 0 0 0 0

1(1/106) 1(1/106) 1(1/106) 2(1.9) 2(1.9) 2(1.9) 3(2.8) 105(99.1) 106(100) 2(1.9) 1(1/16) 226

Note: CIN, cervical intraepithelial neoplasia; ICC, invasive cervical cancer. P2trend, by χ linear-by-linear association; P, Pearson or Fisher's exact test; a dash (–) indicates that there were too few isolates for statistical analysis.

P

Ptrend

(–) (–) (–) (–) (–) (–) 0.138 0.113 1.00 (–) (–)

(–) (–) (–) (–) (–) (–) 0.105 0.113 1.00 (–) (–)

T. Ding et al. / Gynecologic Oncology 119 (2010) 436–443

Fig. 2. Nucleotide sequence variations in the E6 and E7 open reading frames(ORFs) detected in 95 human papillomavirus (HPV) 52 positive Chinese women. HPV52 prototype (GenBank accession number NC_001592) was used as the reference. Nucleotide positions where variations were detected are written across the top. Dashes indicated positions at which no variation was found. Lowercase letters: a silent nucleotide variation; Uppercase letters: a missense variation. The position of amino acid change is stated numerically. The letter preceding this number refers to the reference amino acid and the letter following it refers to its substitution. GenBank accession numbers for sequences reported in this paper are HM004115–HM004117for E6 sequences and HM004118–HM004120 for E7 sequences.

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without C632T (T20I), and the two variants showed a statistically significant positive trend of the association with the severity of neoplasia. Furthermore, we also found that E7 C632T (T20I) and G760A (G63S) were more frequently present in CIN2–3, with significant trends accordant with severity of cervical neoplasia (Ptrend = 0.019, Ptrend = 0.01). It has been revealed that the association between HPV variants and cervical carcinogenesis was relative to geographic region and population differences [31,35]. So, our findings suggest that the correlation between E6/E7 variations especially of E6 A388C (K93N), E7C632T/G760A (T20I/G63S) and cervical neoplasia may be associated with specific geographic regions and population. There have been few studies that investigate the sequence variations in the E6 and E7 genes of HPV 52 and their risk association with the development of cervical neoplasia. Xin et al. detected E6 variations in 46 HPV52-positive Japanese women with cervical neoplasia (24 of CIN 1 and 2, 10 of CIN 3, and 12 of ICC) and found that K93R variant occurred in 45 of 46 samples with HPV52 positive (98%). In accordance with Xin's report, we observed that A379G (K93R) variation occurred in all of the HPV52 positive samples. Additionally, we also observed that simultaneous variations of A378C and A379G, leading to the same variant K93R, in two normal women and one CIN1 patient. The nucleotide variation of G350T was also detected in all of the isolates, which was found in previous Xin's study. We firstly reported variants of G168A (V23M), A378G (K93G), and C366A (N122K) of HPV 52 E6 in the study, but found that they were detected in only one or a few samples and did not do statistical analyses. The E7 variations of HPV 52 were firstly detected in the study, to our knowledge. The 52nd codon of E7 changed from GAC to AGC which led to the 52nd encoded amino acid change from serine to aspartic acid. This mutation may decrease E7 protein phosphorylation rate. It has been indicated that the E7 protein is phosphorylated on serine residues by casein kinase II [36], and a positive association between phosphorylation rate and oncogenic potential has been found [37]. In this study, this mutation was only found in low grade cervical lesions and this may be in accordance with the above findings. We found that C751T and A801G were the two most frequent synonymous mutations at the nucleotide level and occurred in almost HPV52 positive sequenced samples. Additionally, we detected a total of 7 variants of E7, including G592T (D14Y), A599G (Q16R), C622T (T37I), T727G (Y59D), C733T (H61Y), G742A (D64N), and T848G (L99R) in 1 or 2 of 106 HPV52-positive samples. Statistical analyses were not performed because of their too low frequencies. We did not find the prototype of HPV52 E6 and E7 in the study. Taking our results together, we found that a total of 29 new reported variations of HPV 58 and 52, of those, 5 in HPV58 E6, 10 in HPV58 E7, 3 in HPV52 E6, and 11 in HPV52 E7. C632T (T20I) and G760A (G63S) variants in HPV58 E7 were more frequently found in high-grade cervical neoplasia patients, suggesting that they were probably risk factors associated with the development of cervical neoplasia. Although the samples of HPV58 and 52 positive included in the study were larger, this was a cross-sectional study, so any estimation of risk should be interpreted with caution and further investigations are needed. Supplementary materials related to this article can be found online at doi:10.1016/j.ygyno.2010.08.032. Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgments This work was supported in part by the Agency of Science and Technology of Zhejiang Province, Key and High Incidence of Disease Prevention and Control Techniques Special, China (No. 2006C13080); by the National Natural Science Foundation of China (Nos. 30872752 and

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