General Information
This study included 10774 cases of outpatients, inpatients, and physical examination participants undergoing HPV typing tests at the People's Hospital of Anshun City, Guizhou Province, during June 2020 and December 2021, aged 15-86 years, with a mean age of (41±10.94) years. For sampling practice, sexual intercourse was prohibited 24 h before HPV typing and TCT examination, vaginal douching and intravaginal medication was also suspended three days before sampling, neither should the participants be in their menstrual periods. This study was approved by the hospital ethics committee.
HPV typing test
PCR-RDB was used for HPV genotyping detection. 23 HPV subtypes were detected which included HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81, HPV82 , HPV81, HPV82 and HPV83.
TCT test
Cervical sample brush was placed at the cervical orifice and rotated 4-5 turns to collect the cervical exfoliated epithelial cells which were then transferred to the specialized cell preservation solution for transport to the pathology department. Sample bottle was shaken in a shaker for 5 min to pulverize the blood, mucus and non-diagnostic debris, then applied to centrifugation for 3 min at 1200´g, taken out 1 ml sediment on the production rack and centrifuged it again for 3 min(1200´g). The prepared slides were subjected to Pap staining, read by clinical pathologists above the attending physician and the results were reported according to the Bethesda 2014 cervical cell classification system.
Colposcopy
Before the examination, cotton balls pre-soaked in sterile saline was used to wipe the ectocervix secretions, and the uterine cervix was fully exposed with a speculum. Firstly, 3%-5% acetic acid was applied to the cervical orifice for 30s to observe whether there was mosaicism, white epithelium, heterogeneous blood vessels or the appearance of brain gyrus-like changes, and those results were judged positive. Subsequently, the Lugol's iodine was applied on the uterine cervix for 1min. Columnar epithelium and atypical hyperplastic epithelium without staining were defined as the iodine test negative area. Biopsies were taken from the lesions or suspicious lesions and iodine test negative areas.
Histopathological diagnosis
After the target tissues were processed, paraffin sectioning and hematoxylin-eosin staining were performed. The results were interpreted by two clinical pathologists above the attending physician, and were classified according to pathological histological diagnostic criteria: ① normal or chronic inflammation; ② low grade squamous intraepithelial lesions of the uterine cervix (LSIL, i.e. original CIN1) and ③ high grade lesions (HSIL, i.e. original CIN2 and some CIN3); ④ cervical cancer (squamous carcinoma, adenocarcinoma, CC). Those with normal or inflammatory disease were considered negative, with HSIL or higher as an observation.
Immunohistochemical staining
When the diagnosis of pathological tissue biopsy is difficult to grade, immunohistochemical indexes (P16, Ki67) are added to assist in the diagnosis. Test methods were following the reagent manufacturer’s instructions. Results were interpreted as follows: cell nucleus or cytoplasm with orange color was determined as P16 positive; the presence of brownish-yellow particles in the nucleus was determined as Ki67 positive.
Gene sequencing
HPV DNA extraction
DNA was extracted from HPV52 or HPV58 single positive cervical exfoliated cytology specimens using the HPV DNA detection nucleic acid extraction kit, and stored in EP tubes at -80 ℃.
HPV52, HPV58 gene sequencing
HPV52 and HPV58 DNA samples were sent under low-temperature conditions to Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing results were returned for analysis.
Mutation analysis
Sequence mutations and amino acid variations in E6 and E7 regions were analyzed using Snapgene and compared with the reference sequences of HPV52 and HPV58 (HPV52 GenBank ID: NC_001592; HPV58 GenBank ID D90400.1). The E6/E7 gene variant sequences obtained in this study were compared with the reference genome sequences of the E6/E7 variant profile using MEGA11 software with the Align by ClustalW methods, and all the parameters were set to default values. The HPV52 reference genome sequences are: A1: X74481; A2: HQ537739; B1: HQ537740; B2: HQ537743; C1: HQ537744; C1: HQ537746; D: HQ537748 [22]. The HPV58 reference genome sequence are A1: D90400.1; A2: HQ537752; A3: HQ537758; B1: 537762; B2: 537764; C: 537774; D1: 537768; D2: 537770 [22]. The phylogenetic tree was constructed by the neighbor-joining method, with the Kimura2-parameter model selected for parameter settings, Bootstrap value of 1000, and other parameters set to default values. Secondary structure of amino acid sequences were predicted on PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/).
Statistical methods
SPSS 23.0 was used for statistical analysis, and the count data were expressed as the percentage of cases.
Characteristics of HR-HPV infection
A total of 10774 patients were tested for HPV typing, with a positive rate of 20.36% (2,194/10,774). Single infections were predominant, with 1509 cases and a proportion of 14.0% (1509/10774), while 685 cases of mixed infections accounted for 6.36% of total participants (685/10774). Statistically, the distribution of HR-HPV infection subtypes were found to be HPV52 (6.11%), HPV16 (3.59%), HPV58 (3.04%), HPV53 (2.31%), and HPV51 (1.95%) in the order. The distribution of HR-HPV infection is shown in Figure 1.
Distribution of HR-HPV infection in different age groups
Patients undergoing HR-HPV typing were categorized into several groups according to their age. The highest rate of positive HR-HPV infection was in the less-than 20 years old group (36.63%), followed by the greater than 61 years old group (32.30%), the 31-40 years old group (21.63%), the 41-50 years old group (18.27%) and the 51-60 years old group (20.25%). The age-related distribution characteristics of HR-HPV infection are shown in Table 1.
Table 1 Distribution of HPV infection in different age groups
Age
|
Total
|
HR-HPV(+)
|
Precentage(%)
|
<20
|
55
|
20
|
36.36%
|
21-30
|
1831
|
396
|
21.63%
|
31-40
|
3386
|
666
|
19.67%
|
41-50
|
3245
|
593
|
18.27%
|
51-60
|
1743
|
353
|
20.25%
|
>61
|
514
|
166
|
32.30%
|
Note: "+" indicates HPV positive
Characteristics of the distribution of HR-HPV infection in cervical lesions
298 cases were selected to test positive for HPV, and histopathological biopsies were also completed. HPV16 accounted for the highest rate of HR-HPV infection in cervical lesions with a total of 94 cases, including 46 cases of CC and 43 cases of HSIL; 49 cases from HPV52 infection, including 8 cases of CC and 33 cases of HSIL; 34 cases from HPV58 infection, including 5 cases of CC and 23 cases of HSIL; 18 cases from HPV18 infection, including 8 cases of CC and 9 cases of HSIL, 18 cases from HPV33 infection, including 2 cases of CC and 12 of cases HSIL, as shown in Figure 2.
Abnormalities of different HR-HPV in different screening methods
A total of 113 cases of HPV16 positive patients underwent TCT, colposcopy and pathological biopsy examinations at the same time, of which 23 cases were tested positive in TCT, 104 cases were tested positive in colposcopy. Among them, 30 cases were diagnosed with pathological HSIL or above. Meanwhile, 68 cases were found to be HPV52 , while 38 and 33 cases were HPV18 positive and HPV58 positive respectively, and the specific distribution is shown in Table 2.
Table 2 Abnormalities of HPV16, HPV18, HPV52, HPV58 in different testing methods
Subtypes
|
TCT(%)
|
Colposcopy(%)
|
Pathological diagnosis(%)
|
Total(cases)
|
HPV16
|
23(20.35%)
|
104(92.04%)
|
30(26.55%)
|
113
|
HPV18
|
6(15.79%)
|
35(92.11%)
|
8(21.05%)
|
38
|
HPV52
|
24(35.29%)
|
65(95.59%)
|
19(27.94%)
|
68
|
HPV58
|
16(48.48%)
|
31(93.93%)
|
12(36.36%)
|
33
|
Based on the clinical diagnosis results, the same cases while with different types of HPV infections (HPV16, HPV18, HPV52 and HPV58) were selected for further colposcopy, pathological section and immunohistochemical staining examinations, respectively. As seen in Figure 3a, colposcopy results showed that HPV 16, HPV 18, HPV 52 and HPV 58 all can cause chronic cervicitis, different grades of cervical lesions, and CC. The degree of cervix damage caused by HPV infection gradually increases as the lesions get worse. As the gold standard for diagnosis, HE staining can clearly distinguish between chronic cervicitis, different grades of cervical lesions and CC. In chronic cervicitis cases, the cervical epithelium was filled with lymphocytes; in LSIL cases, the heterotypic cells were confined to the epithelium below 1/3; in HSIL cases, the heterotypic cells were greater than 1/3 and accumulated throughout the epithelium but did not break through the basement membrane; in CC cases, the heterotypic cells broke through the basement membrane and infiltrated the interstitium (Figure 3b, 100 times). To further verify the diagnosis results of HE staining and to avoid subjective judgment bias by different reviewers, we selected P16 and Ki67 as auxiliary diagnostic indicators for immunohistochemical staining. The results demonstrated that as the lesions worsened, P16 and Ki67 immunohistochemical staining showed a gradual increase (Figure 3c, d, 100 times).
Gene sequence analysis of HPV52 E6 /E7 regions
The E6/E7 gene regions of 30 HPV52-positive samples were sequenced in this study. The obtained sequences were compared with the HPV52 reference sequence (NC_001592) to find out the mutation sites. The mutation rate of HPV52 E6/E7 gene was 100.00% (30/30) when compared to the reference sequence. The 30 sequencing results were divided into 6 different variant groups (52AS01-52AS06) according to the differential grouping of E6/E7 gene sequence mutations, with the highest frequency of 66.67% (20/30) distributed in the 52AS01 gene mutation group, followed by 20% (6/30) in the 52AS02 gene mutation group . There were 7 single nucleotide mutations in the E6 gene region, among which A125T and A294T were the new found mutation sites. A294T (M65G), A378G (K93G) and A379G (K93R) were non-synonymous mutations, and the rest were synonymous mutations. Sites with highest mutation rates were located in G350T, A379G, and the mutation rate was 100.00% (30/30). On the other hand, there were 3 single nucleotide mutations in the E7 gene region, all of which were synonymous mutations, and the loci with highest mutation frequency were in C751T and A801G, with a mutation rate of 100.00% (30/30). T666C was a new mutation. However, no mutations were found in the amino acid secondary structures of the E6 and E7 sequences as shown in Table 3.
Table 3 Analysis of HPV52 E6/E7 nucleotide mutations and the predicted amino acid secondary structure
|
E6
|
|
E7
|
N
|
Nucleotide
position
|
125
|
294
|
350
|
356
|
378
|
379
|
416
|
|
666
|
751
|
801
|
|
Ref base
|
A
|
A
|
G
|
G
|
A
|
A
|
T
|
|
T
|
C
|
A
|
|
52AS01
|
-
|
-
|
T
|
-
|
-
|
G
|
-
|
|
-
|
T
|
G
|
20
|
52AS02
|
-
|
-
|
T
|
A
|
-
|
G
|
-
|
|
-
|
T
|
G
|
6
|
52AS03
|
-
|
-
|
T
|
-
|
-
|
G
|
C
|
|
-
|
T
|
G
|
1
|
52AS04
|
-
|
-
|
T
|
-
|
G
|
G
|
-
|
|
-
|
T
|
G
|
1
|
52AS05
|
-
|
G
|
T
|
-
|
-
|
G
|
-
|
|
C
|
T
|
G
|
1
|
52AS06
|
T
|
-
|
T
|
-
|
-
|
G
|
-
|
|
-
|
T
|
G
|
1
|
Ref AA
|
R
|
M
|
L
|
G
|
K
|
K
|
I
|
|
D
|
L
|
Q
|
|
Sites
|
8
|
65
|
83
|
85
|
93
|
93
|
105
|
|
38
|
67
|
83
|
|
AA mutations
|
-
|
G
|
-
|
-
|
G
|
R
|
-
|
|
-
|
-
|
-
|
|
Secondary structre
|
C
|
H
|
S
|
H
|
C
|
C
|
C
|
|
C
|
S
|
H
|
|
Note: “-” indicated unmutated sites; “N” indicated samples.
Gene sequence analysis of HPV58 E6/E7 regions
The E6/E7 gene regions of 22 HPV58-positive samples were sequenced in this study. The obtained sequences were compared with the HPV58 reference sequence (D90400.1) using Snapgene software to search for mutation sites. There were 6 cases of HPV58 sequences without mutations, and the mutation rate of HPV58 E6/E7 gene was 72.72% (16/22). 22 sequences were divided into 7 different variant groups (58AS01-58AS06) according to the differential HPV58 E6/E7 gene sequence mutations, with the highest frequency of 37.5% (6/16) distributed in the 58AS01 gene mutation group, followed by 18.75% (3/16) in 58AS02 gene mutation group. There were 3 single nucleotide mutations in the E6 gene region; C321T and A388C were non-synonymous mutations. The site with highest mutation rate was in C307T (62.5%, 10/16).There are eight single nucleotide mutations in the E7 gene region. C632T, G694A, G760A, G761A, T803C, and G806A are nonsynonymous mutations. G744T (81.25%, 13/16) had the highest mutation frequency, while the mutation rate of G694A and G761A were both 37.5% (6/16), as shown in Table 4.
Table 4 Analysis of HPV58 E6/E7 nucleotide mutations and the predicted amino acid secondary structure
|
E6
|
|
E7
|
N
|
Nucleotide
position
|
307
|
321
|
388
|
|
599
|
632
|
694
|
744
|
760
|
761
|
803
|
806
|
|
Ref base
|
C
|
C
|
A
|
|
G
|
C
|
G
|
T
|
G
|
G
|
T
|
G
|
|
58AS01
|
T
|
-
|
-
|
|
-
|
-
|
A
|
G
|
-
|
A
|
-
|
-
|
6
|
58AS02
|
T
|
-
|
-
|
|
-
|
T
|
-
|
G
|
A
|
-
|
-
|
-
|
3
|
58AS03
|
-
|
-
|
C
|
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
2
|
58AS04
|
-
|
-
|
C
|
|
-
|
-
|
-
|
G
|
-
|
-
|
C
|
-
|
2
|
58AS05
|
T
|
-
|
-
|
|
-
|
T
|
-
|
G
|
A
|
-
|
-
|
-
|
1
|
58AS06
|
-
|
-
|
C
|
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
A
|
1
|
58AS07
|
-
|
T
|
C
|
|
A
|
-
|
-
|
G
|
-
|
-
|
C
|
-
|
1
|
Ref AA
|
C
|
S
|
K
|
|
R
|
T
|
G
|
T
|
G
|
G
|
V
|
R
|
|
Sites
|
66
|
71
|
93
|
|
9
|
20
|
41
|
57
|
63
|
63
|
77
|
78
|
|
AA mutations
|
-
|
F
|
N
|
|
K
|
I
|
R
|
-
|
S
|
D
|
A
|
Q
|
|
Secondary structre
|
H
|
H
|
C
|
|
H
|
C
|
C
|
S
|
C
|
C
|
H
|
H
|
|
Note: “-” indicated unmutated sites; “N” indicated samples.
Phylogenetic analysis of HPV52 E6/E7
Phylogenetic tree of HPV52
The phylogenetic tree of HPV52 E6/E7 gene sequences is shown in Fig. 4. The results showed that the mutant mutated sequences of HPV52 E6/E7 genes in Anshun area were all distributed in the B lineage, while no mutations in lineages A, C and D were detected.
Phylogenetic tree of HPV58
The phylogenetic tree of HPV58 E6/E7 gene sequences is shown in Fig. 5. The results showed that the mutated sequences of HPV58 E6/E7 genes in Anshun area were all distributed in the A lineage, while in lineages B, C and D, no mutations were detected.