WO2008142521A2

WO2008142521A2 - Functional assay to investigate unclassified sequence variants of mismatch repair genes

Info

Publication number: WO2008142521A2
Application number: PCT/IB2008/001218
Authority: WO
Inventors: Niels De Wind; Mark Drost
Original assignee: Academisch Ziekenhuis Leiden Leids Universitair Medisch Centrum
Priority date: 2007-05-17
Filing date: 2008-05-15
Publication date: 2008-11-27
Also published as: WO2008142521A3; GB0709445D0

Abstract

The present invention relates to methods of conducting in vitro functional mismatch repair assays, in order to detect whether a mismatch repair gene from a test subject, is incapable of functioning, when the mismatch repair gene comprises a mutation. Such assays are of use in detecting whether or not a subject may be predisposed or have an increased risk of developing cancer, such as hereditary nonpolyposis colorectal cancer (HNPCC).

Description

FUNCTIONAL ASSAY TO INVESTIGATE UNCLASSIFIED SEQUENCE

VARIANTS OF MISMATCH REPAIR GENES Field of the Invention

Background to the Invention

The DNA mismatch repair (MMR) pathway in humans is initiated by heterodimers consisting of homologs of Escherichia coli MutS, which bind directly to single base mismatches and insertion/deletion loops (IDL). In both humans and yeast, MSH2 and MSH6 (MutSα) as well as MSH2 and MSH3 (MutSβ) form the mismatch/IDL recognizing complexes.

Hereditary nonpolyposis colorectal cancer (HNPCC, or Lynch syndrome) is an autosomal dominant hereditary disease caused by germline mutations in MMR genes. Patients suffering from this syndrome inherit mutations in one allele of certain MMR genes, most frequently hMSH2 or hMLHl (W. Abdel-Rahman, J. Mecklin, P. Peltomaki. 2006. The genetics of HNPCC: application to diagnosis and screening. Critical Reviews in Oncology/Hematology 58, 208 - 220). Fortuitous somatic mutation or loss of heterozygosity of the wild-type allele, leaving the cell with a defective MMR system, results in a mutator phenotype. This permits the accumulation of mutations that is thought to drive or contribute to the neoplastic transformation process. The majority of MMR gene mutations in HNPCC patients cause truncations and thus loss of function of the affected polypeptide. However, amino acid alterations comprise a significant proportion of the mutations (-24% of hMSH2, -30% of hMLHl, and -37% of hMSH6) (http://www.insight-group.org/) and the functional consequences of many of these mutations for DNA repair are unclear. A single amino acid substitution, deletion or insertion does not necessarily result in a dysfunctional protein. However, some single amino acid substitutions, deletions or insertions can give rise to partly active, dominant negative, unstable, or non-functional proteins depending on the nature of the individual mutation. Defective MMR caused by missense mutations can be a result of (1) inactivation of enzymatic activity (ATP binding/hydrolysis), (2) defective protein-protein interaction (complex formation), (3) defective protein-DNA binding (mismatch recognition), (4) defective subcellular localization of MMR proteins, (5) altered expression of MMR proteins (stochiometry of MMR complexes), and (6) altered folding or stability of MMR proteins.

As mentioned above, not all mutations result in a dysfunctional protein and unless characterised, it is not easily possible to diagnose those mutations which may contribute to developing cancers, such as HNPCC.

It is amongst the objects of the present invention to provide an easily applicable in vitro assay for detecting whether or not a mutation in a MMR gene leads to a loss or reduction in MMR and as a consequence the potential to develop, for example, cancer such as HNPCC. Summary of the Invention

In a first aspect there is provided a method of detecting whether or not a MMR gene from a subject comprises a mutation which leads to a loss of function or reduction in function of MMR in vitro, comprising the steps of: a) providing a sample of protein expressed by said MMR gene from said subject and contacting this with other necessary reagents so as to allow formation, in normal circumstances, of an enzymatically functional complex comprising said MMR protein; b) contacting said functional complex from a) in the presence of any other necessary co-factors, with a nucleic acid template which comprises at least one base pair mismatch and allowing any repair of said mismatch to occur, wherein the mismatch occurs within a restriction endonuclease recognition site, such that when the mismatch is present, an appropriate restriction endonuclease is substantially incapable of restricting the nucleic acid at said site; and c) detecting any repair of the mismatch, by detecting whether or not the appropriate restriction endonuclease has been able to restrict the nucleic acid template at the restriction endonuclease recognition site.

The sample of protein expressed by the MMR gene may be provided by way of using an in vitro transcription and translation system known in the art, to express protein from the MMR gene. Such systems are available from, for example Ambion, Austin, Texas, US and Promega, Madison, Wisconsin, US and are based on rabbit reticulocyte lysates, wheat germ or bacterial extracts. The mutant MMR gene is regenerated in vitro using conventional PCR-based site-directed mutagenesis technology employing, for example, the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, US). Generally speaking a clone is generated comprising a cloned wild type MMR gene containing at its 5' end a promoter enabling expression in vitro. Optionally, in case the mutated gene is to be expressed in bacterial extracts, a bacterial Ribosome Binding Site may be included. The resulting mutated MMR gene may, for example, be propagated in, and isolated from, Escherichia coli using conventional methods. Alternatively, the mutated MMR gene, fused to DNA sequences that regulate transcription and/or translation, may be regenerated by PCR-based mutagenesis, in a fashion similar to that described above, followed by direct use as a template in in vitro transcription and translation reactions, without prior propagation in E. coli.

The MMR genes which may be tested are the hMSH2, hMSH6, hMSH3, hMLHl, hPMS2, and/or hEXOl genes. Typically, only one of said genes may be tested in each assay, but more than one assay may be carried out in order to test two or more mutated MMR genes. The protein expressed by these genes forms protein complexes which are involved in the MMR and require, for example, nucleotides and ATP to carry out MMR. It is possible to provide all other necessary proteins and necessary co-factors using purified components, see for example Dzantiev, et al, 2004. However, generally speaking it is convenient to use a cell lysate from a cell which is known to be deficient, that is incapable of producing a functional copy of the MMR gene, in order to provide some of the other necessary components, such as the other proteins which are capable of forming the repair complex etc. Additionally proteins or agents which would otherwise degrade or become inactive prior to use of the cell lysate, may be provided in purified form, separately. For example, when testing hMSH2, additional amounts of its partner hMSH6, synthesized in an in vitro expression system from a wild type cloned hMSH6 gene, as described above, is generally required to be added, as hMSH6 in cells that are hMSH2 deficient, quickly degrades within the hMSH2 -negative cell.

The nucleic acid template which comprises said mismatch(es) is typically a DNA template and may be found within a double stranded linear DNA fragment or in a circular form, such as a plasmid. For linear DNA, two substantially homologous single strands may be annealed together, but the strands comprise at least one internal mismatch within a restriction endonuclease site found within the fragment. The sequence may also comprise a suitable site for generating a nick in the template nucleic acid, using a sequence-specific nicking enzyme, such as NtAIwI, Nt.itøvCI, Nb.ZtøvCI, Nb.Bsml. For circular templates, appropriate mismatches and nicks may be generated in accordance with the technique described in Wang & Hays, 2002. Briefly a plasmid is provided which comprises two tandem sites for a sequence- specific nicking endonuclease, like Nt.BstNBl (New England Biolabs, Ipswitch, Massachusetts, US, separated by a region of sequence that includes the restriction endonuclease site. Action by the nicking endonuclease and denaturation removes a single stranded fragment of DNA, which can be replaced by a corresponding oligonucleotide fragment incorporating a mismatch in the region of a restriction endonuclease site to be tested for repair by a MMR system comprising the MMR gene being tested. The latter oligonucleotide may be labelled and can contain covalent modifications such as fluorescent dyes (e.g. Fluorescein, TAMRA, Texas Red^®, JOE, Rhodamine, ROX), as commercially available from, for instance, Eurogentec (Seraing, Belgium). If the MMR gene to be tested comprises a mutation which does not effect functioning of the protein in vitro, the mismatch will be repaired and the restriction endonuclease site generated. However, if the mutation effects functioning of the protein, the MMR will not be carried out, or at a reduced level.

As the present method is based on the repair or otherwise of a restriction endonuclease site, it is a straightforward task to detect whether or not the site has been repaired, by using the appropriate restriction endonuclease, together with a second restriction endonuclease cutting at a predetermined site within the substrate plasmid, and identifying whether or not any restriction of the template at the site of the mismatch has occurred by way of, for example, agarose gel electroporesis. Alternatively, when the mismatch-containing oligonucleotide, used for constructing the mismatch-repair substrate has a label such as fluorescent label attached during synthesis, the labelled diagostic restriction fragment may be detected by analysis on a commercially available DNA analyzer such as the ABI PRISM series (Applied Biosystems, Foster City, US).

The present invention will now be described by way of example and with reference to the following Figures which show:

Figure 1 a shows a plasmid map and nucleotide sequence of the plasmid pJH which is used to generate a template for use in a repair assay;

Figure Ib (panels A - D) shows a schematic representation of how to prepare a suitable nucleic acid template, which comprises a mismatch within a restriction endonuclease site, for use in the present invention; and

Figure 2 shows the results of various tests on a selection of patients who have a mutation in their hMSH2 gene and whether or not such a mutation has an effect on the functioning of the MMR protein. Figure 3 (panel A) shows a fluorescently labelled substrate for MMR, suited for analysis using automated DNA analyzer. The substrate is prepared as in Figure Ib, using a fluorescently-labelled mismatching oligo. (Panel B) Digestion with Mbil and Hindlll generates a fluorescent fragment of 174 basepairs in case no MMR has occurred and a fluorescent fragment of 75 basepairs in case MMR has recreated the Hindlll restriction site. Panel C shows an example of a MMR assay using a fluorescent substrate and MMR-deficient (top) or MMR-proficient (bottom) cell extract. Fragments were analyzed using an automated DNA fragment analyzer.

Figure 4 shows the complete in vitro construction of an unclassified sequence variant of a MMR gene that can directly be used for in vitro expression of protein, followed by an in vitro MMR assay such as described in Figures 2 or 3. Panel A shows the generation, in separate PCR reactions, of two mutant fragments of the MMR gene. After the PCR step, the template is destroyed by adding Dpnl. T7: T7 promoter required for expression in vitro. In a subsequent PCR reaction the two mutation-containing fragments are denatured, annealed, extended and PCR amplified to yield the unclassified sequence variant MMR gene containing all sequences required for direct expression in vitro.

Detailed Description

Example 1 — Generation of mismatched template for use in repair assay

Figure 1 shows in schematic representation how a suitable mismatched template can be formed.

A: The starting plasmid, called pJH (Figure IA and Figure IB, panel A), contains a mutant HmdIII (AAGCCT) and an overlapping Xhol (CTCGAG) site. These are flanked at each side (at approximately 15nt distance) by single strand cutting restriction endonuclease (NtifatNBl) sites, as represented by the two bars (Figure IA) or underlined sequence (Figure IB, panel A).

B: An approximately 30nt single stranded fragment between the Nt&rtNbl sites is removed by action of the Nti&tNBl enzyme (arrowheads in Figure IB panel A), followed by heat denaturation, addition of a excess of complementary oligonucleotide and removal of the ~30nt double and single stranded products by size exclusion chromatography.

C: A kinased oligonucleotide is annealed and ligated into the gap left following removal of the 30nt single stranded fragment, resulting in a mismatch, exemplified by a G. T mismatch, being generated (boxed in Figure IB panel C). Alternatively, a derivative of the oligonucleotide may be used here that contains a covalently attached fluorescent group, enabling to detect the product of the MMR reaction using an automated DNA analyzer (Figure 3). Contaminating homoduplex may be removed at this stage by digestion with Xhol or an isoschizomer of Xhol. Ligated (covalently closed) circular product is isolated by preparative agarose gelelectrophoresis. A further nicking restriction endonuclease Nb.ifo/Ml generates, in this instance, a nick 3' of the mismatch, on the lower strand. This assures that the mismatched nucleotide opposing the annealed and ligated oligonucleotide will specifically be repaired (Figure IB panel C). Example 2 - Assay to test for MMR activity of a subjects hMSH2 gene

The sequence of a suspected HNPCC patient-derived unclassified amino acid change in the hMSH2 gene is obtained from the Clinical Genetics Center at which the respective HNPCC family is screened. Using a cloned wild type hMSH2 gene, preceded by a T7 RNA polymerase promoter, the mutation, together with a linked restriction site (that in itself will not change the amino acid sequence of the gene) enabling facile identification of the mutation, is introduced in hMSH2 employing the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, US). This is followed by introduction of the plasmid in Escherichia coli followed by expansion of the culture and plasmid purification. Alternatively, the mutant hMSH2 gene, including regulatory sequences, may be synthesized as a linear DNA fragment completely in vitro as depicted in Figure 4. The mutated hMSH2 protein encoded by the plasmid, or generated in vitro as a linear DNA fragment, is produced using an in vitro transcription/translation coupled reticulocyte lysate system (Promega, Madison, Wisconsin, US), according to the manufacturer's instructions.

The prepared hMSH2 protein is thereafter incubated with the template prepared in Example 1 (Figure IB panel C) and a cell extract in a buffer including nucleotide cofactors, obtained from MSHi-deficient cells, together with additional wildtype hMSH6, the heterodimeric partner for hMSH2, which also is produced in an in vitro transcription/translation coupled reticulocyte lysate system using a cloned hMSH6 gene as template. MMR reactions contained 100 ng pJΗ (T:G) substrate, 300 μg MSΗ2-deficient cell extract in 20 mM Tris-HCl, pH 7.6; 25 mM KCl; 5 mM MgCl₂; 1.5 mM ATP; 1 mM glutathione; 0.1 mM spermidine; 0.1 mM for each dNTP; total volume of 30 μL. To this, 14 μl reticulocyte lysate containing expressed mutant hMSH2 and expressed wild type hMSH6 in equal amounts, was added. Mixtures were incubated at 37°C for 30 minutes. Reactions were terminated by the addition of 30 μL of Stop Solution (25 mM EDTA, 0.67% sodium dodecyl sulfate and 90 μg/ml proteinase K) and incubated, for 15 min. at 56⁰C. Next, DNA was extracted twice with an equal volume of phenol/chloroform, and precipitated with ethanol. DNA was resuspended in H₂O and digested with 4 units of each HmdIII and Rsal endonucleases at 37⁰C for two hours in 20 μL of Hzwdlll digestion buffer (50 mM Tris-ΗCl, pΗ7.9; 10 mM NaCl; 10 mM MgCl₂; 1 mM dithiothreitol), supplemented with 1.5 μL of a RNase A solution (1 μg/μL, Figure IB panel D). The digested products were separated by electrophoresis in 1% agarose gel in IX TAE Buffer (40 mM Tris- acetate, 2 mM EDTA, pH 8.5). DNA bands were visualized by staining with ethidium bromide, then imaged and photographed with an UV transilluminator (The Gel Doc™ XR System, Bio-Rad, Hercules, California, US.

Correction of T:G mismatches to A:T, in case MMR has occurred, restores a site for Hinάlll, which, in combination with Rsal, cuts the plasmid into 0.5 and 1.5 kBp fragments. In case no MMR has taken place, cleavage by HmdIII will not occur and only a single 2.0 kBp fragment is visible. In the latter case the hMSH2 gene from which the Msh2 protein has been generated is understood to comprise a mutation which can be considered deleterious and capable of potentially leading to development of cancer, such as HNPCC.

The inventors have carried out such assays on a number of presumed HNPCC patients known to have a mutation in their hMSH2 gene, but where it was not known if such a mutation had an effect on protein function. The results are shown in Figure 2 where it can be seen that patients 596, 622, 674 and 697 have a mutation in their hMSH2 gene which leads to a complete loss of function and as such these patients may have a higher risk of developing cancer, such as HNPCC. In addition, patients 380, 675, 834 and 886 have a significant loss of function that may also underlie cancer predisposition. Consequently these patients should be subject to regular clinical investigations, so as to allow early detection and treatment of any cancer and family members that do not carry the mutation are considered to be non-predisposed.

Similar assays have been developed for investigating the functional activity of unclassified sequence variants of the hMSH6 and hMLHl genes.

Claims

1. A method of detecting whether or not a MMR gene from a subject comprises a mutation which leads to a loss of function or reduction in function of MMR in vitro, comprising the steps of: a) providing a sample of protein expressed by said MMR gene from said subject and contacting this with other necessary reagents so as to allow formation, in normal circumstances, of an enzymatically functional complex comprising said MMR protein; b) contacting said functional complex from a) in the presence of any other necessary co-factors, with a nucleic acid template which comprises at least one base pair mismatch and allowing any repair of said mismatch to occur, wherein the mismatch occurs within a restriction endonuclease recognition site, such that when the mismatch is present, an appropriate restriction endonuclease is substantially incapable of restricting the nucleic acid at said site; and d) detecting any repair of the mismatch, by detecting whether or not the appropriate restriction endonuclease has been able to restrict the nucleic acid template at the restriction endonuclease recognition site.

2. The method according to claim 1 wherein the MMR gene is provided by way of an in vitro transcription and translation system.

3. The method according to either of claims 1 or 2 wherein the mutant MMR gene is regenerated in vitro using conventional PCR-based site-directed mutagenesis technology.

4. The method according to any preceding claim wherein the MMR gene(s) to be tested is/are the hMSH2, hMSH6, hMSH3, hMLHl, hPMS2, and/or hEXOl genes.

5. The method according to any preceding claim wherein all other necessary proteins and co-factors for forming an enzymatically functional complex are provided by a cell lysate from a cell which is known to be deficient, that is incapable of producing a functional copy of the MMR gene.

6. The method according to claim 5 wherein additional proteins and/or agents which would otherwise degrade or become inactive prior to use of the cell lysate, are also provided

7. The method according to any preceding claim wherein the nucleic acid template which comprises said mismatch(es) is a DNA template, which is a double stranded linear DNA fragment or in a circular form.

8. The method according to claim 7 wherein the DNA comprises two substantially complementary single strands which strands comprise at least one internal mismatch within a restriction endonuclease site found within the fragment.

9. The method according to claim 8 wherein the DNA also comprises a suitable site for generating a nick in the template nucleic acid, using a sequence- specific nicking enzyme.

10. The method according to any preceding claim wherein the nucleic acid template is labelled with, for example, a fluorescent dye.

12. The method according to any preceding claim wherein any mismatch repair is detected by identifying whether or not any restriction of the template at the site of the mismatch has occurred.

13. The method according to claim 11 wherein identification is carried out by agarose gel electroporesis or by detecting a labelled restriction fragment.

14. The method according to any preceding claim for use in predicting whether or not a subject is predisposed or has an increased risk of developing cancer, such as hereditary nonpolyposis colorectal cancer (HNPCC).