Validation of human papillomavirus genotyping by signature DNA sequence analysis

Department of Pathology, Milford Hospital, Milford, Connecticut, USA.
BMC Clinical Pathology 02/2009; 9:3. DOI: 10.1186/1472-6890-9-3
Source: PubMed

ABSTRACT Screening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing false-positive results from its lower specificity is an important task for the clinical pathologists.
The HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+ primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified equivalent without sample purification or DNA extraction. The nested PCR products were used for direct automated DNA sequencing. A 34- to 50-base sequence including the GP5+ priming site was selected as the signature sequence for routine genotyping by online BLAST sequence alignment algorithms.
Of 3,222 specimens, 352 were found to contain HPV DNA, with 92% of the positive samples infected by only 1 of the 35 HPV genotypes detected and 8% by more than 1 HPV genotype. The most common genotype was HPV-16 (68 isolates), followed by HPV-52 (25 isolates). More than half (53.7%) of the total number of HPV isolates relied on a nested PCR for detection although the majority of HPV-16, -18, -31, -33 -35 and -58 isolates were detected by a single MY09/MY11 PCR. Alignment of a 34-base sequence downstream of the GP5+ site failed to distinguish some isolates of HPV-16, -31 and -33. Novel variants of HPV with less than "100% identities" signature sequence match with those stored in the Genbank database were also detected by signature DNA sequencing in this rural and suburban population of the United States.
Laboratory staff must be familiar with the limitations of the consensus PCR primers, the locations of the signature sequence in the L1 gene for some HPV genotypes, and HPV genotype sequence variants in order to perform accurate HPV genotyping.


Available from: Sin Hang Lee, Sep 15, 2014
1 Follower
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The newly gained knowledge of the viral etiology in cervical carcinogenesis has prompted industrial interests in developing virology-based tools for cervical cancer prevention. Due to the long incubation period from viral infection to developing an invasive cancer, a process whose outcome is influenced by numerous life-style and genetic factors, the true efficacy of the genotype-specific human papillomavirus (HPV) vaccines in cervical cancer prevention cannot be determined for another 30 years. Most HPV DNA test kits designed to replace the traditional Papanicolaou (Pap) smears for precancer detection lack the analytical sensitivity and specificity to comprehensively detect all potentially carcinogenic HPVs and to perform reliable genotyping. The authors implemented the classic nested PCR and Sanger DNA-sequencing technology for routine HPV testing. The results showed a true negative HPV PCR invariably indicates the absence of precancerous cells in the cytology samples. However, 80.5% of single positive HPV-16 tests and 97.3% of single positive HPV-18 tests were associated with a negative or a largely self-reversible Pap cytology. Routine sensitive and reliable HPV type-specific or perhaps even variant-specific methods are needed to address the issues of persistence of HPV infection if a virology-based primary cervical screen is used to replace the Pap cytology screening paradigm.
    12/2014; 6(4):2072-99. DOI:10.3390/cancers6042072
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Knowledge on HPV prevalence and genotype distribution in HSIL and ICC is highly essential for the introduction of an effective vaccination program and appropriate epidemiological monitoring of viral ecology before and after vaccination in Congo. This study aimed to determine the specific-HPV genotypes in HSIL and ICC among women in southwestern Congo. 125 archival paraffin-embedded biopsy collected between 2008 and 2012 and histologically diagnosed were investigated. DNA extraction was performed using the phenol/chloroform method. HPV search was performed by nested-PCR using MY09/MY11 and GP5+/GP6+ consensus primers followed by direct sequencing. The mean age of participants was 44.3 +/- 8.2 years. Overall, HPV prevalence was 89.6% (112/125) with all high-risk genotypes. HPV-DNA was detected in 81.5% (53/65) of HSIL and 98.3% (59/60) of ICC. HPV 16 the most common genotype was detected in 47.1% (25/53) of HSIL and 52.5% (31/59) of ICC. Other types identified were: HPV 33 (22.6%), HPV 18 (15%), HPV 31 (11.3%) and HPV 69 (3.7%) in HSIL, and HPV 33 (28.8%), HPV 18 (11.8%), HPV 31 (5%) and HPV 35 (1.7%) in ICC. Knowing that the ADC accounted for 6.7% (4/60) of ICC cases, HPV 18 was identified in 25% (1/4) of these cases against 75% (3/4) for HPV 16. Our study showed that HPV 16, 33, 18 and 31 were the four most common genotypes in women with HSIL and ICC. These findings indicate that current vaccines against HPV could help to reduce the burden of cervical cancer in Congo.
    BMC Public Health 12/2014; 14(1):1320. DOI:10.1186/1471-2458-14-1320 · 2.32 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cervical cancer is the leading cause of cancer-related death in Africa and the first most common cancer in Gabonese women due to infection of high-risk human papillomavirus (HPV). However, other cofactors such as genetic factors also come into play. A common polymorphism of the p53 codon 72 in exon 4 with two alleles encoding arginine or proline is known at this locus. The homozygous arginine form of this polymorphism has been associated with the development of cervical cancer as an increased genetic risk factor. However, the results are still controversial. This study aims to investigate whether the genotype distribution of p53 codon 72 may be a risk factor for cervical cancer among Gabonese women. Samples from 102 Gabonese women, 31 diagnosed with cervical cancer and 71 healthy controls, were used. HPV detection was done by nested PCR with MY09/11 and GP5+/6+ primers followed by sequencing for HPV genotyping. p53 codon 72 polymorphism determination was performed by allele-specific PCR assay. Viral DNA was detected in 87.1 % of cases and in 54.93 % of control. HPV 16 was the most predominant in cancer and controls cases. The distribution of Arg/Arg, Arg/Pro and Pro/Pro genotypes was 35.5, 51.6 and 12.9 % in the cervical cancer group and 22.5, 62 and 15.5 % in the control group. No significant association was found between polymorphism of p53 itself as well as in combination with HPV16/18 infection and risk of development of cervical cancer among Gabonese women. Thus, the polymorphism of p53 codon 72 in exon 4 does not seem to play a role in the development of cervical cancer among Gabonese women.
    Medical Oncology 12/2014; 32(1). DOI:10.1007/s12032-014-0281-4 · 2.06 Impact Factor