Patent Application
20130084564
(none)
(none)
1. Field of the Invention
A method for detecting or evaluating the risk of developing breast cancer or predisposition to breast cancer. Copy number variations (CNVs) are DNA segments longer than 1 kb for which copy number differences are observed when comparing two or more genomes. The invention results in part from the discovery that a copy number variation containing the RNU2 gene is associated with breast cancer predisposition, possibly by affecting the activity and/or expression of BRCA1, which is a gene associated with breast cancer and for which mutation or diminished expression has been correlated with the development of breast cancer. The inventors have developed a Molecular Combing technique that allows the determination of the number of copies of the RNU2 CNV and therefore assessment of the association between this number and the risk of developing breast cancer.
2. Description of the Related Art
Familial breast cancers account for 5-10% of all breast cancer cases. A mutation in either BRCA1 or BRCA2, the two major genes whose germline mutations predispose to breast and ovarian cancers, is suspected when there is a strong family history of breast or ovarian cancer, for example, when the disease occurs in at least three first or second-degree relatives such as sisters, mothers, or aunts.
If the function of the protein encoded by BRCA1 is impaired, for example, by a gene mutation in the coding region, then damaged DNA is not repaired properly and this increases the risk of cancer.
Similarly, BRCA2 encodes a protein involved in DNA repair and certain variations or mutations in these gene are associated with a higher breast cancer risk.
When a patient is found to be at risk of familial breast cancer, then molecular genetic testing may be offered and carried out if the patient desires it. Molecular testing is offered to women with breast and/or ovarian cancer belonging to high-risk families. When a BRCA1 or BRCA2 mutation is identified, predictive testing is offered to all family members >18 years old. If a woman tests negative, her risk becomes again the risk of the general population. If she tests positive, a personalized surveillance protocol is proposed: it includes mammographic screening from an early age, and possibly prophylactic surgery. Chemoprevention of breast cancer with anti-estrogens is also currently tested in clinical trial and may be prescribed in the future. However, for 80% of the tested families no mutations are identified and all women of the negative families go on being monitored regularly though with a less stringent protocol than do carriers of known mutations to BRCA1 or BRCA2. Moreover, though frame shift, nonsense or splice site mutations are the most frequent BRCA1 mutations, they do not explain all the BRCA1 linked families.
The numerous mutations identified in BRCA1/2 (>2,000 different ones) are mostly truncating mutations occurring through nonsense, frame shift, splice mutations or gene rearrangements (Turnbull, 2008). However, no mutation was identified in BRCA1 or BRCA2 in 80% of the tested breast cancer families and no other major predisposing gene seems to exist (Bonaïti-Pellié, 2009). This represented a significant problem for diagnosing genetic predisposition to breast cancer in a large proportion of these families.
As explained below, the inventors investigated copy number variations (CNVs) associated with the RNU2 gene which may lie in close proximity to BRCA1 and were able to show that other mechanisms besides mutations in. BRCA1 or BRCA2 may account for increased predisposition to breast and ovarian cancer in some of these families.
CNVs represent copy number changes involving a DNA fragment of 1 kilobase (kb) or larger (Feuk, 2006). They are found in all humans and mammals examined so far and along with other genetic variations like single-nucleotide polymorphisms (SNPs), small insertion-deletion polymorphisms (indels), and variable numbers of repetitive sequences (VNTR) are responsible for human genetic variation. Characterizing human genetic variation has not only evolutionary significance but also medical applications, as this may elucidate what contributes significantly to an individual's phenotype, and provides invaluable tools for mapping disease genes.
The extent to which CNVs contribute to human genetic variation was discovered a few years ago (Iafrate, 2004; Sebat et al., 2004; Hurles, 2008) and CNVs have thus gained considerable interest as a source of genetic diversity likely to play a role in functional variation. Indeed, they represent approximately 10% of the genome (Conrad, 2007; Redon et al., 2006).
In most cases, CNVs result from the duplication or the deletion of a sequence and are bi-allelic, i.e., only two alleles are present in the population. It has been shown recently that common CNVs that can be typed on existing platforms and that are well tagged by SNPs are unlikely to contribute greatly to the genetic basis of common human diseases (The WTCCC, 2010). However, 10% of the CNVs are multi-allelic: they can result from multiple deletions and duplications at the same locus and frequently involve tandemly repeated arrays of duplicated sequences (Conrad, 2010). The highly multi-allelic CNVs are not tagged by SNPs. Furthermore, the greater the number of alleles found in the general population, the more difficult it is to type them. However, almost all of the reported associations of CNVs to diseases involve multi-allelic ones (Henrichsen, 2009).
Whatever the content of the repeated sequence, the CNVs may influence the expression of distant genes, either through the alteration of the chromatin structure or through the physical dissociation of the transcriptional machinery by cis-regulators (Stranger et al., 2007).
Recent investigations in mice have suggested that the effect of CNVs on the expression of flanking genes could extend up to 450 kb away from their location (Henrichsen, 2009). Moreover, long CNVs (>50 kb) would affect the expression of neighboring genes to a significantly larger extent than small CNVs. In 2006, Merla et al. showed that not only hemizygous genes that map within, the microdeletion that causes Williams-Beuren syndrome show decreased relative levels of expression, but also normal-copy neighboring genes (Merla, 2006). Furthermore, fascioscapulohumeral muscular dystrophy (FSHD) has been directly related to the copy number of a polymorphic repeat: D4Z4. In patients, a partial deletion of the repeats (copy number <8) causes the loss of a nuclear matrix attachment site, found initially between the D4Z4 repeats and the neighboring genes. This absence is suspected to be responsible for the activation of these genes (Petrov, 2006).
In 1984, Van Arsdell et al. described the RNU2 CNV as a nearly perfect tandem array of a 6 kb basic repeat unit containing the 190 bp-long gene coding for the snRNA U2, RNU2-1 (1984). The basic unit has been sequenced in 1995 (Accession number: L37793), as well as the flanking junctions (Pavelitz, 1995). By pulsed field; gel electrophoresis (PFGE), this locus has been found to be highly polymorphic, the number of copy measured in 50 individuals varying between 5 and >30 (Liao, 1997). This CNV maps to a major adenovirus 12 modification site on 17q21 (Lindgren, 1985), and it has also been shown that this locus lies approximately 120 kb upstream of the BRCA1 gene (Liu, 1999).
The inventors have identified and characterized copy number variations (CNVs) that can explain BRCA1 inactivation and predisposition to breast or ovarian cancer associated with BRCA1 inactivation. These include large rearrangements in genomic sequences, in particular, a recurrent duplication that is one the most frequent mutations (Puget, 1999) and a recombination hot spot involving the BRCA1 pseudogene (Puget, 2002). They investigated whether BRCA1/2 could be inactivated in some instances through alternative mechanisms, such as chromatin alteration mediated by a copy number variation (CNV) and confirmed the presence 120 kb upstream of BRCA1 of a multi-allelic and highly polymorphic CNV described in the literature, despite its absence in the current human genome assembly (Build 37). The structure of the RNU2 CNV located close to BRCA1 was characterized by various means including extraction of relevant data in available databases and by PCR, FISH and sequencing analyses. These investigations determined the correct sequence for the basic unit of RNU2 CNV, its correct length, and showed that actual sequence had a 6.1 kb length in comparison to the published sequence described as having a length of 5.8 kb.
Moreover, the inventors employed Molecular Combing to confirm the location of CNVs upstream BRCA1 and to study the polymorphic characteristics of this segment of the genome. Molecular Combing, as well as materials and protocols for performing Molecular Combing, are known and are incorporated by reference to U.S. Pat. Nos. 5,840,862; 6,054,327; 6,225,055; 6,248,537; 6,265,153; 6,294,324; 6,303,296; 6,344,319; 6,548,255; 7,122,647; 7,368,234; 7,732,143; and 7,754,425.
By analyzing five individuals, it was shown that the size of the RNU2 CNV could extend up to 300 kb, which corresponds to the size range of CNVs known to modify the expression of neighboring genes.
Furthermore, they used quantitative PCR (q-PCR) to measure the number of repeats in seven individuals in order to correlate this number with breast cancer risk. Four of these individuals were also analyzed by Molecular Combing and the inventors showed that there is a good correlation between the RNU2 copy number estimated by these two techniques. They then studied the influence of the RNU2 CNV locus on breast cancer susceptibility: more than 2,000 samples were tested by qPCR, the positive correlation between number of copies and risk of cancer was confirmed.
The discovery of an association between BRCA1 associated copy number variations, such as those comprising the RNU2 segment, and cancer risk provides new methods and tools for assessing the risk of predisposition to cancer, especially breast and ovarian cancer.
Based on these discoveries, products and methods useful for detecting the presence of, or the location of, one or more genes or of one or more sequences of RNU2, especially RNU2 copy number variants associated with BRCA1 on the same DNA molecule were developed.
Products according to the invention may constitute one or more molecules reacting with RNU2 CNV DNA or DNA sequences flanking the RNU2 CNV DNA. These products include probes that bind to RNU2 CNV sequences or its flanking sequences and can identify sequences outside of the BRCA1 or BRCA2 genes associated with a genetic predisposition to breast or ovarian cancer.
Methods according to the invention includes those which attach DNA molecules containing RNU2 CNV DNA to a combing surface, combing the attached molecules, and then reacting the combed DNA molecules with one or more labeled probes that bind to RNU2, RNU CNV, or flanking sequences.
Moreover, these methods can extract information in at least one of the following categories:
(a) the position of the probes on combed DNA,
(b) the distance between probes on the combed DNA, and/or
(c) the size or length of the probes along the combed DNA (e.g., the total sum of the sizes, which makes it possible to quantify the number of hybridized probes).
The location of an RNU sequence, the number of RNU2 sequences and the length of RNU2 copy number variations may be determined from this information. This information may also be used to detect or locate specific kinds of RNU2 sequences such as polymorphic RNU2 sequences.
In the Molecular Combing technology according to the invention a “combing surface” corresponds to a surface or treated surface that permits anchorage of the DNA and DNA stretching by a receding meniscus. The surface is preferably a flat surface to facilitate readings and examination of DNA attached to the surface and combed.
“Reaction between labeled probes and the combed DNA” encompasses various kinds of immunological, chemical, biochemical or molecular biological reactions or interactions. For example, an immunological reaction can comprise the binding of an antibody to methylated DNA or other epitopes on a DNA molecule. An example of a biochemical or chemical reaction or interaction would include binding a molecule, such as a protein or carbohydrate molecule, to one or more determinants on a DNA molecule. An example of a molecular biological interaction is hybridization of a molecule, such as a complementary nucleic acid (e.g., DNA, RNA) or modified nucleic acid probe or primer, to a DNA substrate. There may also be mentioned, as examples, DNA-DNA chemical binding reactions using molecules of psoralen or reactions for polymerization of DNA with the aid of a polymerase enzyme. A hybridization is generally preceded by denaturation of the attached and combed DNA; this technique is known and will not be described in detail.
The term “probe” designates both mono- or double-stranded polynucleotides, containing at least synthetic nucleotides or a genomic DNA fragment, and a “contig”, that is to say a set of probes which are contiguous or which overlap and covers the region in question, or several separate probes, labeled or otherwise. “Probe” is also understood to mean any molecule bound covalently or otherwise to at least one of the preceding entities, or any natural or synthetic biological molecule which may react with the DNA, the meaning given to the term “reaction” having been specified above, or any molecule bound covalently or otherwise to any molecule which may react with the DNA.
In general, the probes may be identified by any appropriate method; they may be in particular labeled probes or alternatively non-labeled probes whose presence will be detected by appropriate means. Thus, in the case where the probes are labeled with methylated cytosines, they could be revealed, after reaction with the product of the combing, by fluorescent antibodies directed against these methylated cytosines.
The elements ensuring the labeling may be radioactive but will preferably be cold labelings, by fluorescence for example. They may also be nucleotide probes in which some atoms are replaced.
The size of the probes can be of any value measured with an extensive unit that is to say such that the size of two probes, is equal to the sum of the sizes of the probes taken separately. An example is given by the length, but a fluorescence intensity may for example be used. The length of the probes used is between for example 5 kb and 40-50 kb, but it may also consist of the entire combed genome.
Advantageously, in the method in accordance with the invention, at least one of the probes is a product of therapeutic interest that will interact with RNU2 CNV DNA.
Preferably, the reaction of the probe with the combed DNA is modulated by one or more molecules, solvents or other relevant physical or chemical parameters.
In general, while the term “genome” is used within this text; it should be clearly understood that this is a simplification; any DNA or nucleic acid sequence capable of being attached to a combing surface is included in this terminology. In addition, the term “gene” will sometimes be used indiscriminately to designate a “gene portion” of genomic origin or alternatively a specific synthetic or recombinant “polynucleotide sequence”.
Specific embodiments of the invention include the following.
An isolated or purified polynucleotide that binds to an RNU2 polynucleotide sequence, an RNU2 CNV (copy number variation sequence), or a sequence flanking the RNU2 CNV or that is useful as primer for the amplification of an RNU2 polynucleotide sequence or RNU2 CNV or for a sequence lying between BRCA1 and an RNU2 sequence or a sequence flanking a RNU2 CNV.
The isolated or purified polynucleotide of Embodiment 1 that is selected from the group consisting of L1 (nt 20-542) (SEQ ID NO: 27), L2 (nt 731-1230) (SEQ ID NO: 28), L3 (nt 1738-2027) (SEQ ID NO: 29), L4 (nt 3048-3481) (SEQ ID NO: 30), L5 (nt 3859-5817) (SEQ ID NO: 31), R1 (nt 1-485) (SEQ ID NO: 32), R2 (nt 1288-1787) (SEQ ID NO: 33), R3 (nt 2075-4237) (SEQ ID NO: 34), R4 (nt 4641-5022) (SEQ ID NO: 35), R5 (nt 5391-5970) (SEQ ID NO: 36), R6 (nt 6702-7590) (SEQ ID NO: 37), C1 (SEQ ID NO: 60), C2 (SEQ ID NO: 61), C3 (SEQ ID NO: 62) and C4 (SEQ ID NO: 63); or a polynucleotide that hybridizes under stringent conditions (e.g., remains hybridized after washing in 0.1×SSC and 0.1% SDS at 68° C.) with said isolated or purified polynucleotide or its full complement.
The isolated or purified polynucleotide of Embodiment 1 that is a probe specific for RNU2 CNV selected from the group consisting of SEQ ID NOS: 27-36 and 37.
The isolated or purified polynucleotide of Embodiment 1 that is a primer selected from the group consisting of SEQ ID NOS: 1-26 and 52-59.
The isolated or purified polynucleotide of Embodiment 1 that is a primer useful for directed amplification by qPCR of the RNU2 CNV region selected from the group consisting of L1Fq (SEQ ID NO: 38), L1Rq (SEQ ID NO: 39), and Taqman L1 (SEQ ID NO: 42).
A kit for detecting the genetic predisposition of developing a breast or an ovarian cancer comprising primers for amplification of DNA corresponding to RNU2 CNV region, probes specific for RNU2 CNV, and/or optionally primers and/or probes specific for BRCA1 gene expression.
A method of detecting the number of copies of an RNU2 sequence in a sample containing an RNU2 copy number variant (CNV) comprising contacting the sample with one or more probes that identify an RNU2 CNV sequence of interest, and determining the number of sequences based on the characteristics of probe binding to the sequence of interest.
The method of Embodiment 7, where the sample contains several genomic DNA molecules with potentially different numbers of sequences of an RNU2 copy number variant and potentially sequences of an RNU2 copy number variant within different genomic regions and where the number of sequences is determined independently for each genomic DNA molecule and optionally where the number of sequences is determined independently for RNU2 copy number variants from different regions
The method of Embodiments 7 or 8, where the sample contains human genomic DNA from a single individual and where the number of sequences determined represents the average number of sequences on the two alleles of the genomic region of interest.
The method of Embodiments 7 or 8, where the sample contains human genomic DNA from a single individual and where the number of sequences is determined independently for the two alleles of the genomic region of interest
The method of Embodiments 7 to 10, where the sample is prepared for array-based Comparative Genomic Hybridization (aCGH) prior to contacting immobilized probes suitable for determining the copy number of the RNU2 CNV in aCGH procedures.
The method of Embodiments 7 to 10, where the sample is prepared for DNA microarray procedures prior to contacting immobilized probes suitable for determining the copy number of the RNU2 CNV in DNA microarray procedures.
The method of Embodiments 7 to 10, where the sample is prepared for Fluorescence in Situ Hybridization (FISH) procedure prior to contacting the probes and where the probes are suitable for determining the copy number of the RNU2 CNV in FISH procedures.
The method of Embodiments 7 to 10 where the sample is prepared for Southern blotting procedure prior to contacting the probes and where the probes are suitable for specific hybridization on the DNA molecules containing the RNU2 CNV in Southern blotting procedures and where the number of sequences is determined based on the size of DNA molecules hybridized to the probes.
The method of Embodiments 7 to 10 where the sample is subjected to molecular combing prior to contacting the probes and the probes are suitable for determining the copy number of the RNU2 CNV in molecular combing procedures.
The method of Embodiment 15, wherein determining the number of RNU2 sequences comprises determining (a) the position of the probes, (b) the distance between probes, or (c) the size of the probes (the total sum of the sizes which make it possible to quantify the number of hybridized probes).
The method of Embodiment 15, wherein said probe is selected from the group consisting of L1 (nt 20-542) (SEQ ID NO: 27), L2 (nt 731-1230) (SEQ ID NO: 28), L3 (nt 1738-2027) (SEQ ID NO: 29), L4 (nt 3048-3481) (SEQ ID NO: 30), L5 (nt 3859-5817) (SEQ ID NO: 31), R1 (nt 1-485) (SEQ ID NO: 32), R2 (nt 1288-1787) (SEQ ID NO: 33), R3 (nt 2075-4237) (SEQ ID NO: 34), R4 (nt 4641-5022) (SEQ ID NO: 35), R5 (nt 5391-5970) (SEQ ID NO: 36) and R6 (nt 6702-7590) (SEQ ID NO: 37); or a polynucleotide that hybridizes under stringent conditions (e.g., remains hybridized after washing in 0.1×SSC and 0.1% SDS at 68° C.) with said isolated or purified polynucleotide or its full complement.
A method of detecting the number of copies of an RNU2 sequence in a sample containing an RNU2 copy number variant (CNV) comprising contacting the sample under conditions suitable for amplification of all or part of the RNU2 CNV; amplifying all or part of the RNU2 CNV in the sample using DNA polymerases and; determining the number of sequences based on the characteristics of the amplified product or products.
The method of Embodiment 18, wherein said primers are selected from the group consisting of SEQ ID NOS: 1-26 and 52-59 or a primer useful for directed amplification by qPCR of the RNU2 CNV region selected from the group consisting of L1Fq (SEQ ID NO: 38), L1Rq (SEQ ID NO: 39), and Taqman L1 (SEQ ID NO: 42).
A method for assessing the risk of developing cancer or a predisposition to cancer in an individual comprising determining the average length or number of copies in an RNU2 CNV in this individual; optionally correlating the said length or copy number with a risk or predisposition to cancer; optionally correlating the said length or copy number with expression of a BRCA1 gene associated with said RNU2 CNV on a DNA molecule; and/or optionally determining a risk or predisposition to cancer when the RNU2 CNV reduces the expression of BRCA1.
A method for assessing the risk of developing cancer or a predisposition to cancer in an individual comprising determining the lengths or numbers of copies in an RNU2 CNV in several alleles in this individual; optionally correlating the said lengths or copy numbers with a risk or predisposition to cancer; optionally correlating the said lengths or copy numbers with expression of a BRCA1 gene associated with said RNU2 CNV on a DNA molecule; and/or optionally determining a risk or predisposition to cancer when the RNU2 CNV reduces the expression of BRCA1.
The method of Embodiment 20 or 21, wherein a risk or predisposition to cancer is positively correlated with RNU2 CNV length or RNU2 copy number.
The method of Embodiment 20 or 21, wherein a risk or predisposition to cancer is determined by comparison of the lengths or copy numbers of an RNU2 CNV in the sample with a reference value established as being a minimum value characteristic of a risk or predisposition to cancer.
The method of Embodiment 23 wherein the reference value is established as the minimum average value characteristic of a risk or predisposition to cancer and wherein this reference value is preferably comprised between 40 and 150 copies or the corresponding length (more preferably between 70 and 125 copies or the corresponding length).
The method of Embodiment 23 wherein the reference value is established as the minimum value for a single allele characteristic of a risk or predisposition to cancer and wherein this reference value is preferably comprised between 20 and 150 copies or the corresponding length (more preferably between 50 and 125 copies or the corresponding length and more preferably between 35 and 100 copies or the corresponding length)
The method of Embodiment 20 or 21, wherein expression of a BRCA1 gene is determined by detecting mRNA transcribed from said gene.
The method of Embodiment 20 or 21, wherein expression of a BRCA1 gene is determined by detecting the presence of a polypeptide expressed by the BRCA1 gene.
The method of Embodiment 20 or 21, wherein the presence of said polypeptide is detected by one or more antibodies that bind to a normal or to a mutated BRCA1 polypeptide.
The method of Embodiments 20 to 28, wherein said cancer is ovarian cancer or breast cancer.
Use of molecular combing to detect the presence or absence of RNU2 CNV or the number of copies of RNU2 in a DNA molecule containing BRCA1.
Use of molecular combing to detect the presence or absence of genetic abnormalities at an RNU2 locus associated with BRCA1, wherein an RNU2 abnormality is defined as a structure of the RNU2 locus found at a higher frequency in a subject having a lower level of BRCA1 expression than the level of BRCA1 expression of a normal subject.
Use of molecular combing to detect the predisposition of developing ovarian or breast cancer by identification of BRCA1 and RNU2 CNV genes or copies thereof in a sample.
A method of determining a genetic predisposition to breast or ovarian cancer comprising screening DNA from a subject or amplified from a subject by Molecular Combing using one or more probes that bind to RNU2, RNU2 copy number variants, polynucleotide flanking RNU2 or RNU2 copy number variants, or sequences between RNU2 and BRCA1,
determining a genetic predisposition to breast or ovarian cancer when the location, length or number of RNU2 copies differs from those of subjects not genetically predisposed to breast or ovarian cancer.
The method of Embodiment 33, wherein said subject does not have a BRCA1 or BRCA2 gene variant associated with predisposition to breast or ovarian cancer.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.
A single RNU2 sequence is found on chromosome 17 reference sequence in an annotated sequence named LOC100130581. The proposed organization of the RNU2-BRCA1 region deduced from data published in the literature is presented in
The human reference assembly for chromosome 17 found in Build 36 annotated the RNU2 locus in the unplaced NT—113932.1 contig. This contig was based on a single unfinished RP11-570A16 BAC sequence (AC087365.3). The AC087365.3 sequence contains sixteen unassembled contigs. Part or the entire L37793 sequence is found in all but contigs 1 and 16, and 10 copies of RNU2 (called the RNU2-1 gene) are found in total. The TMEM106A gene and the end of the NBR1 gene are found in contig 1. The left junction of the RNU2 CNV, sequenced in 1995 by Pavelitz et al. (1995), is found at the end of contig 15, while the right junction is found at the beginning of contig 16. However, in Build 37 (dated from March 2009) this BAC was removed from the assembly so the RNU2-1 gene was no longer found there.
Currently, the RNU2-2 gene localized on chromosome band 11q12.3 is considered to be the functional gene for snRNA U2. While RNU2-4P (also known as RNU2P) (288 bp long) has been assigned to chromosome 17 (41,464,596-41,464,884), but is referred to as a pseudogene. Furthermore, this sequence is present only once in an annotated sequence of 7.6 kb named LOC100130581 (
The LOC100130581 and L37793 sequences are partly homologous and both can be amplified from genomic DNAs. Using the NCBI Blast algorithm Blast2Seq, six regions of homology were found between the LOC10030581 and the L37793 sequences, amounting to a total of 2142 bp (
The inventors undertook a PCR analysis in order to determine if two different regions exist in the genome whose sequence correspond respectively to LOC100130581 and L37793 or if these latter correspond to the same region that has been inaccurately sequenced in one instance. An attempt was made to amplify the LOC100130581 sequence from genomic DNA using primers R1F and R6R (
The same approach was used with the L37793 sequence. The L1F and L5R primers allowed the amplification from genomic DNA of the expected 5.8 kb fragment (
Both of these highly homologous sequences were amplified from genomic DNAs, so FISH analyses were performed to determine their localization. FISH analysis was first performed using the RP11-100E5 BAC (AC087650) containing the LOC10013058 sequence, as verified by PCR amplification (data not shown). This BAC was found localized on chromosome band 17q21 (
FISH analysis was then performed using the approximately 5.8 kb PCR product obtained with primers L1F and L5R. A green signal was visualized with the labeled fragment (
L37793 Contains an Alu Repeat Omitted in Previous Data
To determine the complete sequencing of L37793, sequencing of PCR fragments covering, the entire fragment was performed and the sequences were assembled manually. The obtained sequence is 6,153 nt long (SEQ ID NO: 64), roughly 300 nt, longer than the published 5,834 nt sequence. Sequence comparison shows that an Alu repeat, located at position 1,711 in our sequence, was omitted from the sequence published for L37793.
The LOC100130581 Sequence Leads to an Incomplete Visualization of the RNU2 CNV.
In order to determine if LOC100130581 was repeated and was close to BRCA1, Molecular Combing technology was used. This technology allows the visualization of fluorescent signals obtained by in situ hybridization of probes on combed DNA where DNA fibres are irreversibly attached, stretched, and aligned uniformly in parallel to each other over the entire surface of a-vinylsilane-treated glass. The physical distance measured by optical microscopy is proportional to the length of the DNA molecule and is at the kilobase level of resolution (2 kb).
The barcode developed by Genomic Vision for the BRCA1 gene provided a panoramic view of this gene and its flanking regions, which covers TMEM, NBR1, LBRCA1 (pseudo-BRCA1), NBR2 and BRCA1. This approach has been used for identifying BRCA1 large rearrangements in French breast cancer families (Gad et al., 2002). Since each probe size is known, this can be used to estimate the size of new signals, such those of any RNU2 repetitions.
To avoid non-specific hybridization, PCR fragments specific to the LOC sequence and containing no more than 300 bp of repeated sequences (Alu, LTRs . . . ) were designed to be used as probes and named R1, R2, R3, R4, R5 and R6 (
Only the Phusion and Fermentas polymerases led to reproducible amplification of the R2 to R6 regions, giving rise to fragments of the expected size: 500 bp for R2; 2.2 kb for R3; 400 bp for R4, 500 bp for R5, and 900 bp for R6 (
Two combed DNAs provided by Genomic Vision (referred as donor 1 and donor 2) were analyzed. For both donors, only the end of the BRCA1 barcode developed by Genomic Vision (covering TMEM, NBR1 and LBRCA1) was used.
For donors 1 (
Probe R5 comprises the RNU2 gene, therefore it was concluded that it was highly likely that the RNU2 CNV lies upstream of the BRCA1 gene. However, the red dots upstream of BRCA1 don't have an uniform size and the spacing between these dots was not homogeneous. Whether they result from partial or perfect hybridization of R4, R5 or R6 probes cannot be determined at this stage. To determine if the LOC100130581 sequence is indeed repeated, PCR analyses were conducted from genomic DNA using inversely oriented primer pairs: R6F-R2R, R6F-R1R, R5F-R1R. These pairs will only lead to amplification if part or the entire LOC100130581 sequence is repeated. No band was obtained with any of the Taq polymerases and the primer pairs used (data not shown), suggesting that LOC100130581 or even part of this sequence is not repeated in the human genome. These data suggest that the signals visualized by molecular painting are likely to result from cross-hybridization of the R probes with the homologous L37793 sequence (
The L37793 Sequence is the Repeat Unit of the RNU2 CNV.
Inversely oriented primers were designed specific to the RNU2-1 sequence, ReRNU2F/R, which allow the amplification of a fragment only if the RNU2 sequence is repeated at least once (
Molecular Combing technology was employed in order to confirm that L37793 is close to BRCA1 and to determine the number of repeats in a few individuals. Five regions specific to L37793 and containing no more than 300 bp of repetitive sequences have been defined: L1, L2, L3, L4 and L5 (
The L37793 sequence was then studied by Molecular Combing on three individuals. For the analysis of the DNA of the first individual, the L5 probe was labeled in green, while the L1 to L4 probes were labeled in red (
For the analysis of the two other individuals, the L1 to L4 probes were labeled in blue while the L5 probe was labeled in red. Using these probes, a repeated sequence could also be observed upstream of BRCA1, but only repeated red dots are visible. For individual 2, seven signals were found on the scanned slide (
Finally, for individual 3, 45 signals showing the CNV upstream of BRCA1 have been measured, giving an average size for red dots of 2.15 kb±0.63 (out of 230 analyzed) and an average size for the gap between these points of 4.30 kb±2.21 (
For these three individuals, the average size of the gap between the end of the BRCA1 bar code (the TMEM106A gene) and the beginning of the CNV was 30.31 kb±5.30. The distance between the end of the TMEM106A gene and the beginning of the BRCA1 gene being 90 kb, the CNV would be at an average distance of 120 kb upstream of BRCA1.
The highest relative copy number ratio was identified in the patient diagnosed with breast cancer at the earliest age. A real-time q-PCR approach was used to determine the copy number ratio of the L1 region of the L37793 sequence versus the single-copy NBR1 gene in seven individuals belonging to high-risk breast cancer families and for whom no BRCA1/2 mutation was found. The relative copy number (RCN) was determined in three independent experiments, each performed in triplicate. The ratios obtained are all different, varying from 20 to 53, which suggest that each individual of this small series has a different total copy number of the L37793 sequence (Table 1).
Molecular combing analysis performed on the DNA of four individuals out of the seven analyzed by q-PCR showed that there was a good correlation between the global copy number estimated by these two techniques (
Table 1. Age of diagnosis of breast cancer, mean relative copy number (RCN) quantified by qPCR and global copy number (GCN) quantified by molecular combing of the CNV RNU2 for seven individuals belonging to high-risk breast cancer families. The mean RCN were obtained on three independent experiments, each one made in triplicate. SD: standard deviation. The global copy numbers (GCN) were obtained by molecular combing on four independent hybridization experiments, by adding the mean value for each allele. ND: not done.
Based on the results reported herein, it appears that in some breast cancer families, the length of the RNU2 CNV correlates with risk of breast cancer and this correlation may be associated with impairment of BRCA1 expression. Recently, CNVs have been described to represent a great portion of the genome, and some studies have shown that they can influence the expression of neighboring genes (Henrichsen, 2009).
Characterization of the Region Upstream of BRCA1.
Initially, the current human chromosome 17 assembly was studied and compared with the data found in the literature. Discrepancies were identified, which induced the inventors to investigate the content of the region upstream of BRCA1 through a PCR approach. Several PCR amplification problems have been met when trying to amplify the L37793 and LOC100130581 sequences, probably due to their content. Indeed, amplification of DNA fragments containing Alu and LTR sequences, as well as dinucleotides repeats, is often difficult, especially when performed from genomic DNA and in the case of long sequences (larger than 1 kb). Thus, several TAQ polymerases and cycling conditions have been tested in order to be able to obtain sound and reproducible results, which was achieved for both regions and gave rise to PCR fragments with the expected sequence. It was concluded from these experiments that both regions exist in the genome.
On the other hand, amplification of the R1 region was not accomplished and the smear that was systematically obtained has not been explained, especially as not only the R1-R6 region could be amplified from genomic DNA, but R1 could also be readily amplified from a BAC.
FISH analyses localized both the L37793 sequence and the RP11-100E5 BAC containing LOC100130581 at 17q21. The fact that a strong signal was obtained with an approximately 6 kb probe (corresponding to the L37793 sequence), while FISH is usually performed with probes at least 100 kb-long, indicates that this sequence is repeated. This was further confirmed as it was managed to PCR amplify fragments from the L37793 sequence with primers in reverse orientation and given the results obtained by Molecular Combing. Taken together, these results show that the L37793 sequence is indeed the repetitive unit of the RNU2 CNV.
By Molecular Combing, it was also confirmed that this CNV was located about 120 kb upstream of BRCA1. Therefore, it was concluded that the current human genome assembly for chromosome 17 was inaccurate. The sequence of the region upstream of BRCA1 is not reliable probably because of the difficulty to assemble the sequence of the RP11-570A16 BAC (AC0087365.3). This latter, although containing the left and right junctions of the CNV and 10 copies of the RNU2 gene, has been left unassembled and removed from the most recent version of the assembly. Although a new assembly has been proposed in September 2011 (AC0087365.4), the proposed data still does not allow locating or characterizing the RNU2 CNV correctly, as the assembly is still only partial and excludes most data relative to the repeated sequence.
This shows that the assembly of the human genome relies only on bioinformatics methods and that data from the literature are not integrated. As a result, essential data such as the presence of a CNV in close proximity to a major cancer predisposing gene are at the moment omitted in the human genome reference. As genotyping and expression microarrays are fundamentally dependent upon the reference genome for array probe design, this implies that a small but possibly highly relevant fraction of the human genome has not been adequately analyzed at present.
Manual assembly of the 16 contigs of the RP11-570A16 BAC was performed in order to determine the genetic content of the region lying between TMEM106A and the RNU2 CNV and to place the CNV sequence within the BRCA1 upstream region. Primers have been specifically designed at the end and the beginning of each contig. PCR amplification could then be performed using random primer pairs and sequencing of the PCR products will place the contigs into order. This allowed us to propose a final assembly (
Using this new assembly, we designed additional probes for the RNU2 locus, flanking the repeat array in close proximity (a few kb) to its ends. These probes were obtained by PCR on the RP11-570A16 BAC or on total human genomic DNA. Primer sequences were based on contigs in AC0087365.3 as well as NW—926828.1 and NW—926839.1 and the expected sizes were obtained for PCR fragments, which were partially sequenced, with the expected results. Probes C3 (predicted sequence: SEQ ID NO: 62; expected size: 7078 nt) and C4 (predicted sequence: SEQ ID NO: 63; expected size 5339 nt) hybridize between the RNU2 CNV and the LOC100130581 sequence, while probes C1 (predicted sequence: SEQ ID NO: 60; expected size: 4857 nt) and C2 (predicted sequence: SEQ ID NO: 61; expected size 4339 nt) hybridize between the RNU2 CNV and the BRCA1 gene.
The content of this BAC suggests that the RNU2 CNV lies approximately 30 kb upstream of TMEM106A, and approximately 70 kb downstream of the LOC100130581 sequence (Suspected localization of the CNV at position 41,400 K,
It is not possible to know at this stage whether the LOC100130581 and the L37793 sequences share the same evolutionary origin. However, it is possible that the LOC100130581 sequence was previously part of the RNU2 CNV, and has been separated from the rest of it because of massive LTR insertions between them. Indeed, the 70 kb that is suspected to lie between the LOC100130581 sequence and the CNV are mainly constituted by LTR sequences according to the human genome assembly and the NW-926839.1 contig. So it could be that after this insertion, the LOC100130581 sequence was no more submitted to selection, explaining the divergence between them. The RNU2 CNV locus has been described to be highly submitted to selection: all the repetitions are identical (Liao, 1997). To date, no function has been associated with the LOC100130581 sequence, its fixation in human populations can be due to genetic drift, a major process in human genome evolution. Thus it is proposed that the RNU2 sequence present in LOC100130581 is a pseudogene as are other RNU2 sequences present on others chromosomes.
Design of Tests for RNU2 CNV
Reliable information about the sequence of the region located upstream of the CNV is required for improving the Molecular Combing technique. For example, a new set of probes needed to be designed in order to frame the repeats to ensure that the entire CNV is visualized. The inventors therefore designed the C1/C2 and C3/C4 set of probes described above and the position of theses probes relatively to the RNU2 CNV was precisely determined. Besides, a precise size assessment for a single repeat unit is required if the number of copies is to be deduced from the total size of the repeat array. In this way, a more accurate count the number of copies can be obtained.
Molecular Combing is a highly powerful technique for analyzing multiallelic CNVs constituted by short repeats, as it can lead to the determination of the number of repeats much more precisely than with PFGE.
With the inventors' characterization of the RNU2 CNV and its genomic region, Molecular Combing tests can be designed to determine the number of copies with improved accuracy. A test based on Molecular Combing scan be based on sets of probes including:
Probes that allow the determination of the number of copies of RNU2 sequence within the RNU2 CNV may be, for example, probes that hybridize on the RNU2 repeat units and that allow the identification of individual copies of the repeat unit, thus allowing to count them. We have successfully used probes L1, L2, L3, L4 and L5, with probes L1, L2, L3, L4 labeled in red and L5 in green: each repeat unit appears as a pair of successive red and green spots. Counting the number of pairs of red and green spots is a direct assessment of the number of repeat units. Using probes that hybridize over part of the repeat unit may also allow counting individual units, as they would appear as distinct spots. Typically, if the probes cover a 3 kb stretch in the repeat unit, the 3 kb-probe would be readily detected, while the 3 kb-gap separating two successive probes would allow to tell the probes apart and thus count them. We have successfully used probes L4 and L5, both labeled in red. Each repeat unit appears as a red spot and two consecutive repeat units can readily be told apart, and thus the number of repeat units can be directly counted.
Alternatively, the number of repeat units may be deduced from the total length of the repeat array, since the length of a single repeat unit is known. This can be achieved with probes hybridizing on the RNU2 repeat units, by measuring the total length formed by the succession of these probes. If the probes hybridize over only part of a repeat unit, it may be required to correct the total length by adding the length of the non-hybridized part before dividing by the length of a repeat unit. Alternatively, the measurement may be made between one end of the first repeat unit and the same end of the last repeat unit, thereby measuring the length of all but one repeat units,
The length of the repeat array may also be obtained using probes flanking both sides of the repeat array. Provided the position of these probes relative to the extremities of the repeat array are known with sufficient precision, the length of the repeat array can be obtained from the distance between the flanking probes, corrected for the space between the probes and the actual extremities of the repeat array. We have used the distance between extremities of the C1/C2 probe, on one side, and the C3/C4 probe, on the other side, closest to the repeat array. Since there is a ˜5 kb gap between the C1/C2 probe and the repeat array and a ˜2 kb gap between the C3/C4 probe and the repeat array, 7 kb is subtracted from the measured distance to obtain the length of the repeat array. In such a setup, it is possible to completely omit probes hybridizing on the repeat units themselves, although such probes allow the confirmation of the presence of the repeat units.
Obviously, several assessment procedures for the number of copies may be combined, e.g., for increased accuracy or for confirmation of one method with another one.
Probes that allow the distinction of RNU2 CNVs from the region of interest from potential homologous sequences may be readily designed using known procedures for Molecular Combing, since we have established with sufficient precision the assembly of the region including the RNU2 CNV. Indeed, probes from the region surrounding the RNU2 CNV may be designed and their specificity for this region confirmed in Molecular Combing experiments. Such confirmation experiments may involve hybridizing the intended probes simultaneously with the probes forming the barcode for BRCA1 which we have described previously, and confirming that they hybridize in the expected position relatively to the BRCA1 gene.
Furthermore, if it is deemed necessary to confirm the location of the RNU2 CNV in proximity to the BRCA1 gene or to another gene (e.g., because the expression of such a gene may be modulated by the RNU2 CNV only if it is sufficiently close), probes specific for the BRCA1 gene or other genes of interest may be hybridized simultaneously with the probes used for the measurement of the RNU2 CNV. Probes specific for the BRCA1 gene or other genes of interest are previously published or may be designed using procedures known to the man skilled in the art.
Probes that allow to assess whether a signal for an RNU2 CNV is intact may be used to allow sorting out partial RNU2 CNV repeat arrays, e.g. when the DNA fiber was broken in the CNV during sample preparation. Such probe sets typically comprise probes flanking the RNU2 repeat array on both sides. If only probes from one side are present in a signal, it may be assumed that the fiber Was broken and the measurements may be excluded from e.g. calculations of average size. Since fiber breakage occurring in the gap between the flanking probes and the repeat array, leaving the repeat array intact, would lead to exclusion of useful data, this gap should be as small as possible so the probability of this is minimal. Thanks to our detailed assembly of the region, we have been able to design the C1/C2 and C3/C4 probes so the gap is only a few kb, and the probability of breakage within the gap practically insignificant.
The stretching factor, i.e., the ratio between the nucleotide length of a sequence and its physical length on the combed slide as measured by microscopy, is on average. 2 kb/μm, but it may vary from slide to slide (with an estimated standard deviation of 0.1-0.2 kb/μm). The accuracy of the determination of the number of copies within a CNV may be improved by correcting for this variation, especially if the copy number is deuced from the total length of the RNU2 CNV repeat array. Measurements of one or several sequence(s) of known size(s) on the same slide may be used to calculate the stretching factor.
As can be expected in such widely polymorphic CNV, most individuals have two alleles of the RNU2 CNV with different copy numbers. In a single molecule test such as Molecular Combing test, the size of the two alleles may be determined independently. Procedures for the determination of average sizes for the two alleles independently have been published elsewhere and are readily adaptable by the man skilled in the art.
Using a probe set consisting of: L4, L5 (red), C1, C2 (green), C3, C4 (blue), and probes from the previously published BRCA1 barcode, we have been able to accurately measure the size of individual alleles in 9 individuals with global copy numbers ranging from 37 to 244 as determined by qPCR (
The number of copies in a RNU2 CNV may also be estimated by FISH procedures. Indeed, although the spatial resolution of FISH does not allow the direct measurement of the repeat array or the counting of individual repeat units, the fluorescence intensity of a probe hybridizing on the repeat units is strongly correlated with the number of copies. For example, we have analyzed samples from two individuals presenting high copy numbers as determined by qPCR (approximately 160 and 220 copies, respectively), using the entire sequence of a repeat unit as a probe. We have been able to show that the first individual had two alleles with comparably high copy numbers, since the fluorescence of the probes on both chromosomes 17 were comparable, while the second had one allele with a high copy number and another with a low copy number, as reflected by the much stronger fluorescence intensity of the probe on one of the chromosome. Further adaptation of FISH procedures to establish an estimation of copy numbers in absolute or relative terms are readily accessible to the man skilled in the art.
PCR-based techniques do not allow one to determine the number of repeats on each allele. However, these techniques are usually fast and relatively inexpensive and both types of techniques may be used in complementary manner. We have developed quantitative PCR procedures that allow a reliable assessment of the number of copies of the RNU2 sequence in a sample. This was made possible because we could unambiguously characterize the sequence of the repeat unit in the CNV, allowing for example to evade interference with the LOC100130581 sequence. We therefore designed primers and a probe that are specific to the sequence of the repeat unit, avoiding any homology with the LOC100130581 sequence. We have found this to work best when measurements were performed in duplicate, using the RNAse P gene as a calibrator. Based on the now precisely characterized sequence of the repeat unit, the man skilled in the art could readily derive other qPCR primers and probes for the RNU2 CNV, as well as design tests based on other common quantitative techniques such as array-based comparative genomic hybridization (aCGH), etc.
Number of Copies of the RNU2 CNV Repeat and Level of Expression of the BRCA1 Gene.
The number of copy has been reported in the literature to vary between five and >30. Nothing is known about the degree of heterogeneity of the population regarding this CNV. However, among the little number of individuals that we analyzed in the initial study, the CNV RNU2 has been shown to be highly polymorphic, as the number of repeats seemed to differ for each allele. One individual presented at least 53 copies, which means that this CNV can thus extend up to at least 300 kb. Work is underway to analyze breast cancer families with no mutation in BRCA1/2 with the objective of identifying families with a very large number of repeats. In the course of this larger-scale study, the highest copy number count for a single allele to date is 175 copies (roughly 1 Mb). It has been described that long stretches of repeated, sequences can promote heterochromatisation and it is hypothesized that in certain conditions, heterochromatic regions can spread over the neighboring regions. We therefore propose that a very large number of repeats in the case of the CNV RNU2 could lead to BRCA1 transcriptional silencing.
However, in the case of the FSHD syndrome, Petrov et al showed that the deletion of some D4Z4 repeats have repercussion on chromatin structure, merging two chromatin loops and bringing the contracted repeats and neighboring genes into the same transcriptional environment (Petrov, 2006). Thus another objective is the identification of families with an unusually low number of repeats.
The results obtained to date concerning the copy number ratio of the CNV RNU2 in seven individuals belonging to high-risk breast cancer families seem to indicate that this ratio is higher in individuals who developed a breast cancer before the age of 40. At the present time, multi-allelic CNVs are poorly studied: only a small number of them are present in the actual human genome assembly. As it has been shown very recently that bi-allelic CNVs are unlikely to contribute greatly to the genetic basis of common human diseases (The WTCCC, 2010), it is important now to test the implication of multi-allelic CNVs. These have not been included yet in genome-wide association studies as they are not tagged by SNPs and because they are difficult to type. The characterization of the CNV RNU2 and its association with BRCA1 and the use of Molecular Combing provide valuable tools to analyze and evaluate predisposition to cancer, especially breast cancer.
Number of Copies of the RNU2 CNV Repeat and Risk of Cancer.
1,183 breast cancer cases and 1,074 controls have been studied by duplex qPCR, allowing to determine the global copy number distribution in the general population, and in a population of index cases. The mean global copy number was 52.53 [51.33-53.72] for index cases and 50.24 [49.11-51.30] for controls and statistical tests show a significant difference in mean copy number and distribution of copy numbers. In the general population, the distribution followed a Gaussian curve: the minimum was 12 copies, and the maximum was 154 copies. Interestingly, in the index cases population, the maximum was 243 copies. RNU2 copy number resulted to be higher than the maximum in the control population in 3 index cases. Familial information has been obtained for index cases with a high RNU2 global copy number. Individuals with high copy number were often found in the same family associated with cancer, validating our hypothesis of high RNU2 copy number being associated with high risk of developing breast and potentially other cancer. Since a high RNU2 copy number has been also found individuals affected by skin cancer, an association between the RNU2 CNV and other cancer forms cannot be excluded.
Materials
Human lymphoblastoid cell lines have been established by Epstein-Barr virus immortalization of blood lymphocytes at the diagnostic laboratory at the Centre Léon Bérard. Lymphoblastoid cells of control individuals (not diagnosed with cancer) were cultivated in RPMI 1640 medium (Sigma-Aldrich), supplemented with 1% penicillin-streptomycin and 20% fetal bovine serum (Invitrogen). Genomic DNA was extracted with the NucleoSpin kit (Macherey-Nagel). The seven individuals analyzed by q-PCR all belong to high-risk families and have a personal history of breast cancer (see Table 1 for age at diagnosis). They have furthermore tested negative in a BRCA1/BRCA2 diagnosis test aiming at detecting point mutations and genomic rearrangements.
Two bacterial artificial chromosomes (BACs) containing regions of interest of chromosome 17, have been purchased: RP11-100E5 (Invitrogen) (AC087650 accession number, which corresponds to nt: 41,406,987-41,576,514 of NC—000017.10), containing the LOC100130581 sequence (
Sequence Data Analyses
The human chromosome 17 assembly used for sequence analyses is referred as NC—000017.10 in the NCBI database. It is the latest assembly (March 2009) and contains 81,195,210 bp. The BRCA1 gene sequence coordinates are: 41,196,314-41,277,468. The L37793 sequence, deposited in the NCBI database in 1995 by Pavelitz et al (1995), is 5,834 bp long. The LOC100130581 sequence, found on the chromosome 17 assembly (41,458,959-41,466,562) is 7,604 bp long. Blast analyses were performed using the BlastN algorithm parameters on NCBI.
PCR Amplification and Probe Synthesis
PCR and long-range PCR were performed in 20 μL reactions. Cycling conditions were chosen according to the polymerase and the length of the sequence to amplify. The following four Taq polymerases were used: Taq Platinium, Invitrogen (94° C. for 2 min, 35 cycles of (94° C. for 20 s, Tm° C. for 30 s, 72° C. for 1 min/kb), 72° C. for 7 min), PfuUltra II Fusion HS DNA Polymerase, Agilent (92° C. for 2 min, 30 cycles of (92° C. for 10 s, Tm-5° C. for 20 s, 68° C. for 30 s/kb, 68° C. for 5 min), Phusion High-Fidelity DNA Polymerase, Finnzymes (98° C. for 30 s, 30 cycles of (98° C. for 10 s, Tm° C. for 20 s, 72° C. for 30 s/kb), 72° C. for 7 min), Long PCR Enzyme Mix, Fermentas (94° C. for 2 min, 10 cycles of (96° C. for 20 s, Tm° C. for 30 s, 68° C. for 45s/kb), 25 cycles of (96° C. for 20 s, Tm° C. for 30 s, 68° C. for 45s/kb+10 s/cycle), 68° C. for 10 min, in the presence of 4% DMSO for amplification longer than 5 kb). PCR products were analyzed on a 1.5% agarose gel containing 0.5× Gel Red (Biotium) with 1 μg of the MassRuler DNA Ladder Mix (Fermentas).
Primers were designed with the Primer3 v.0.4.0 software (http://_frodo.wi.mit.edu/primer3/) to allow the amplification of 5 or 6 regions of the L37793 or LOC100130581 sequences respectively and synthesized by Eurogentec. These regions were chosen in order to include no more than 300 bp of repeat sequences (such as Alu or LTR sequences), according to the Repeat Masker software (http://_www.repeatmasker.org/cgibin/WEBRepeatMasker). Primer sequences and temperature of annealing are the following:
A primer pair has been designed to specifically amplify the RNU2 coding region:
An inversely oriented primer pair has been designed to specifically amplify a RNU2 repetition:
A primer pair has been designed to amplify one region flanking the RNU2 CNV, in between the CNV and LOC100130581:
Primer pairs have been designed to amplify 3 regions flanking the RNU2 CNV, in between the CNV and BRCA1:
Based on our new assembly of the RP11-570A16 BAC, we designed new primer pairs for the amplification of probes flanking the RNU2 CNV region, between the CNV and LOC100130581:
Based on our new assembly of the RP11-570A16 BAC, we designed new primer pairs for the amplification of probes flanking the RNU2 CNV region, between the CNV and BRCA1:
The probes for Molecular Combing were synthesized by PCR using genomic DNA (50 ng) for the L37793 sequence and for the C3 and C4 sequences, DNA extracted from the RP11-100E5 BAC (0.05 ng) for the LOC100130581 sequence or DNA extracted from the RP11-570A16 BAC (0.03 ng) (see Materials) for the S1, S2, S3, S4, C1 and C2 sequences. PCR products, except for fragment S1, S2, S3 and S4, have been cloned within the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions. Competent TOP10 bacteria were transformed with 1 ng of this vector, and cultivated on solid LB medium containing Ampicilin and X-gal. Blue colonies were grown overnight in liquid LB Amp medium. Plasmid DNAs were extracted with Mini or Midi NucleoSpin Plasmid kit (Macherey-Nagel), and verified by sequencing (Cogenics).
Probe Sequences
After amplification and sequencing, the probe sequences for L37793 and LOC100130581 were determined.
Probes C1, C2, C3 and C4 were partially sequenced, which confirmed the following predicted sequences, based on AC0087365.3, NW—926828.1 and NW—926839.1:
The full sequence for the RNU2 repeat unit was determined by sequencing the entire PCR fragment obtained with L1F and L5R:
Random Priming
The six probes obtained for LOC100130581, the five probes for L37793 and the four probes flanking the RNU2 CNV were labeled by random priming, simultaneously with the last three probes of the BRCA1 barcode (elaborated by Genomic Vision). Probes that have been labeled with the same fluorochrome were coupled. 200 ng of each probe were incubated during 10 minutes at 100° C. with 1× random primers (Bioprime), and then cooled at 4° C. during 5 minutes. Klenow enzyme (40 U) and dNTP 1× (2 mM dGTP, 2 mM dCTP, 2 mM dATP, 1 mM dTTP) were then added to this solution. Depending on the chosen emission color, dNTPs 1 mM coupled with biotin (for red emission), digoxygenin (for blue emission), or Alexa-488 (for green emission) were also added. These mixes were incubated overnight at 37° C., and the priming reaction was then stopped with EDTA 2·10−2 mM pH 8.
Molecular Combing
DNA molecular combing was performed at the Genomic Vision company, according to their protocol: for preparing DNA fibres of good quality, lymphoblastoid cells (GM17724 and GM17739) were included in agarose blocks, digested by an ESP solution (EDTA, Sarcosyl, Proteinase K) and then by β-agarase in a M.E.S solution (2-N-Morpholino-Ethane sulfonique 500 mM pH 5.5). This DNA solution was incubated with a silanized coverslip, which was then removed from the solution with a constant speed of 300 μm/sec. This protocol allows' maintenance of a constant DNA stretching factor of 2 kb/μm (Michalet et al., 1997).
Hybridization
One tenth of each random priming mix was precipitated during one hour at −80° C. with 10 μg of Human. Cot1 DNA, 2 μg herring sperm DNA, one tenth of volume of AcNa 3M pH 5.2 and 2.5 volumes of Ethanol 100%. After centrifugation during 30 minutes at 4° C. and at 13.500 rpm, the supernatant is discarded and the pellet is dried at 37° C. and dissolved with hybridization buffer (deionized formamid, SSC (salt sodium citrate) 2×, Sarcosyl 0.5%, NaCl 10 mM, SDS 0.5%, Blocking Aid). 20 μL of the mix are laid on a coverslip with combed DNA, denatured at 95° C. during 5 minutes, and incubation is then performed overnight at 37° C.
Probe Detection
Hybridized coverslips were washed three times (3 minutes each) with formamide—SSC 2×, and three times with SSC 2×. Coverslips were then incubated 20 minutes at 37° C. in a wet room with the first reagents: Streptavidine-A594 for Biotin-dNTP (1), Rabbit anti-A488 antibody for Alexa-A488-dNTP (2), and Mouse anti-Dig AMCA antibody for Digoxygenin-dNTP (3). Coverslips were washed with three successive baths of SSC 2×-Tween20 1%. Similarly, coverslips were incubated with the second reagents: Goat anti-streptavidine biotinylated antibody (1), Goat anti-rabbit A488 antibody (2) and Rat anti-mouse AMCA antibody (3). Coverslips were washed and incubated with the third reagents: Streptavidine A594 (1), and goat anti-rat A350 antibody (3). Coverslips were dehydrated with three successive baths of ethanol (70-90-100%). Observation was conducted with epifluorescent microscope (Zeiss, Axiovert Marianas), coupled with a CCTV camera (Photometrix Coolsnap HQ), with the 40× objective and the Zeiss Axovision Rel4.7 software. Signals were studied with ImageJ (available from NHI) and Genomic Vision home-made softwares (Jmeasure224).
Number of copies was determined by counting the number of signals corresponding to a repeat unit or by measuring the length of the repeat array (between probes C1/C2 and C3/C4 when these probes were included) and dividing by the length of one repeat unit.
Fluorescent In Situ Hybridization
FISH studies were performed using probes amplified from genomic DNA for L37793 or using one BAC (RP11-100E5) and using the 17 subtelomeric probe. In this latter case, DNA was extracted according to standard techniques. Both probes were labeled using the nick translation method.
q-PCR Amplification of the RNU2 CNV Copy number for the RNU2 CNV was determined using the TaqMan detection chemistry. Primers were designed to specifically amplify a 72 bp-amplicon from the L1 region of the L37793 sequence and showing no homology with LOC100130581: L1Fq 5′-GAGGTGCAGGTAGTATAAGCCATT-3′ (SEQ ID NO: 38), and L1Rq 5′-GAGCCACGATGCTTGGAC-3′ (SEQ ID NO: 39). To account for possible variation related to DNA input amounts or the presence of PCR inhibitors, a reference gene, NBR1, was simultaneously quantified in separate tubes for each sample with primers NBR1F 5′-TGGTACAGCCAACGCTATTG-3′ (SEQ ID NO: 40) and NBR1R 5′-ATCCCATACCCCAATGACAG-3′ (SEQ ID NO: 41) (size of the amplicon: 92 bp). The sequences of the TaqMan probes are: Taqman L1 5′-ACGGAACGCACAGGAGCAGAG-3′ (SEQ ID NO: 42), NBR1 5′-CTGCCTGCTGCTCAGAGATGATCTT-3′ (SEQ ID NO: 43).
Primers and probes were synthesized by Eurofins MWG Operon. Optimal primer and probe concentrations were determined according to the TaqMan Gene Expression Master Mix protocol (Applied Biosystems). They were for NBR1, 500 nM and 100 nM respectively, and for L1 50 nM for both primers and probe. PCR reactions were performed on a Applied Biosystems Step One Plus Real-Time PCR System Thermal Cycling Block in a 20 μL volume with 1× TaqMan Gene Expression Master Mix, optimal forward and reverse primers concentration, optimal. TaqMan probe concentration, 25 ng of DNA. The cycling conditions comprised 10 min at 95° C., and 40 cycles at 95° C. for 15 sec and 60° C. for 1 min.
For each experiment, the mean Ct value for L1 and NBR1 was determined in triplicate. The ΔC
ΔC
The relative copy number (RCN) was calculated using the following formula: RCN=ΔC
Alternatively, an improved protocol was used for qPCR:
Copy number for the RNU2 CNV was determined using the TaqMan detection chemistry. Primers were designed to specifically amplify a 72 bp-amplicon from the L1 region of the L37793 sequence and showing no homology with LOC100130581: L1Fq 5′-GAGGTGCAGGTAGTATAAGCCATT-3′ (SEQ ID NO: 38), and L1Rq 5′-GAGCCACGATGCTTGGAC-3′ (SEQ ID NO: 39). To account for possible variation related to DNA input amounts or the presence of PCR inhibitors, a reference gene, RNaseP, was simultaneously quantified in separate tubes for each sample with the primers and probes from Applied Biosystems. The sequence of the TaqMan probe for L1 is: Taqman L1 5′-ACGGAACGCACAGGAGCAGAG-3′ (SEQ ID NO: 42).
Primers and probes were synthesized by Eurofins MWG Operon, except for RNAse P which was purchased from Applied Biosystems. RNaseP was used at 1× concentration, L1 at 50 nM concentration and L1F and L1R at 100 nM each. PCR reactions were performed on a Applied Biosystems Step One Plus Real-Time PCR System Thermal Cycling Block in a 20 μL final reaction volume with, 1× TaqMan Gene Expression. Master Mix, the above-mentioned concentration for primers and probe and 20 ng of DNA. The cycling conditions comprised 2 min at 50° C. followed by 10 min at 95° C., and 40 cycles at 95° C. for 15 sec and 60° C. for 1 min.
For each experiment, the mean Ct value for L1 and RNAse P was determined in duplicate. The ΔC
ΔC
ΔΔC
The relative copy number (RCN) was calculated using the following formula: RCN=2(−ΔΔCt).
The ranges disclosed herein include all subranges and intermediate values.
Each document, patent, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety, especially with respect to the specific subject matter surrounding the citation of the reference in the text. However; no admission is made that any such reference constitutes background art and the right to challenge the accuracy and pertinence of the cited documents is reserved.
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/493,010, filed Jun. 3, 2012, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61493010 | Jun 2011 | US |
