Full HPV typing by a single restriction enzyme

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Abstract

Background

Restriction fragment length polymorphism (RFLP) methods for genotyping genital human papillomavirus (HPV) are considered labor consuming and constrained by the reduced set of restriction enzymes capable of detecting specific mutations. However, we think that these methods have not taken full advantage of the high diversity of the known restriction enzymes.

Objective

We have set out to find the best restriction enzyme for HPV typing.

Study design

An extensive search for enzymes was carried out by combining statistical methods and database information. The search maximized the discrimination between high- and low-risk types by examining the sequence of the L1 gene flanked by primers MY09/11. Different electrophoretic resolutions and two variations of the RFLP method were considered.

Results

HpyCH4V is the best enzyme for discriminating between risk types. Moreover, HpyCH4V generates different patterns for virtually all the HPV types. The typical pattern consists of two or three fragments, which facilitates typing in mixed infections. The typing of a set of clinical samples confirmed the expectations.

Conclusions

This result illustrates the possibilities of statistical methods to exploit the high diversity of restriction enzymes in order to classify samples in a pre-established hierarchy of types for which DNA sequences are known.

Introduction

Epidemiological and genetic studies have shown that human papillomavirus (HPV) causes many forms of genital dysplasia and neoplasia (Baseman and Koutsky, 2005, Bosch et al., 2002, Wang and Hildesheim, 2003). Over 40 different HPV types have been described as infecting the anogenital tract (de Villiers et al., 2004) but only some of them are considered to be associated with lesions with a significant risk of malignant progression (Muñoz et al., 2003). Therefore, the classification of types causing infections is clinically important for the identification of patients at risk of developing cervical lesions and cancer.

A variety of methods have been developed for HPV typing using either type-specific probes or PCR amplification with consensus or specific primers. The performance and reproducibility of these tests have been widely discussed (Hubbard, 2003, Molijn et al., 2005). Our effort is centered on methods based on restriction fragment length polymorphism (RFLP) analysis of PCR amplifications using consensus primers (Lungu et al., 1992, Wang et al., 1999). Two main difficulties are associated with these methods. The first one is related to the use of consensus primers for PCR amplification: different HPV genotypes are amplified and detected with different efficiencies by the same set of primers. Moreover, the amplification of a particular genotype in a mixed infection is dependent on the combination of genotypes present in the original sample. In the recent past, there has been a considerable effort to design new sets of primers, which amplify a broader spectrum of HPV types (Gravitt et al., 2000, Jacobs et al., 1997, Kleter et al., 1999, Sasagawa et al., 2000, van Ham et al., 2005). The second difficulty corresponds to the fact that RFLP patterns are often very complex, particularly in mixed infections, and the resolution of fragment sizes does not allow distinction between different genotypes. Additionally, the presence of spurious bands can make the pattern difficult to read.

The power of discrimination between genotypes by RFLP analysis depends on the region of the HPV genome amplified by PCR and the particular set of restriction enzymes used to generate fragments. The segment of the L1 region amplified by either the set of primers MY09/11 (Manos et al., 1989) or the redesigned set PGMY09/11 (Gravitt et al., 2000) has proven to be a valid tool for identifying genotypes. The level of variation of this sequence allows the discrimination of all the genotypes and it has been itself a reference for the characterization of types and variants (see Bernard, 2005). The restriction enzymes used to reveal this variation are some of the most common in laboratories: BamHI, DdeI, HaeIII, HinfI, PstI and RsaI (Lungu et al., 1992, Meyer et al., 1995, Naqvi et al., 2004). It seems as if the election of enzymes has been made paying more attention to their accessibility than to their suitability for the task. We think that this approach does not take advantage of the potential utility of the RFLP methods for HPV genotyping. Here we describe a computer-aided search for restriction enzymes that maximizes the discrimination of genotypes for the L1 region flanked by the set of primers MY09/11. The aim is to find enzymes that produce patterns of fragments that clearly identify the high-risk HPV types.

Section snippets

Statistical evaluation of restriction enzymes

A file with the recognition sequences of 140 type-II restriction enzymes (Table 1) was compiled from the Rebase database (Roberts et al., 2005). A second file with the DNA sequences of 41 HPV types for the region defined by primers MY09/11 was also compiled from the GenBank databases (Benson et al., 2002). The GenBank accession numbers of the HPV types used as references are given in Table 2. The file was split into two sections according to the risk classification of the different HPV types

Typing by unlabeled RFLPs

The computer screening of 140 restriction enzymes was performed for resolutions of the electrophoresis system ranking from virtually 0% (fragments with different lengths are always identified as different fragments) to 5% (two fragments differing by less than 5% are considered to be the same fragment). The threshold of detection of restriction fragments was set to 80 bp: fragments under this size were not considered for discrimination of HPV types. Under these settings, five restriction enzymes

Discussion

The extensive computer search concluded with the finding of a single restriction enzyme that allows the detection of high-risk HPV types present in a sample. Furthermore, HpyCH4V generates different patterns of fragments for almost all the HPV types infecting the anogenital tract. Most of these patterns are clearly discernible even with electrophoresis systems with low discrimination power and prone to perturbations of the mobility due to the particular sequence of the fragments.

Until now, RFLP

Acknowledgements

This work was supported by grant BMC2003-03022 from the Ministerio de Ciencia y Tecnología of Spain. We are also grateful to Biozell Diagnóstico Molecular for technical assistance.

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