US 2013 O130246A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0130246A1 BENSMON et al. (43) Pub. Date: May 23, 2013 (54) METHODS FOR THE DETECTION, (22) Filed: Oct. 31, 2012 VISUALIZATION AND HIGH RESOLUTION PHYSICAL MAPPING OF GENOMIC Related U.S. Application Data REARRANGEMENTS IN BREAST AND OVARAN CANCER GENES AND LOC (60) Provisional application No. 61/553,906, filed on Oct. BRCA1 AND BRCA2 USING GENOMIC 31, 2011. MORSE CODE IN CONJUNCTION WITH MOLECULAR COMBING Publication Classification (71) Applicants: Aaron BENSIMON, Anthony (FR): (51) Int. Cl. Maurizio Ceppi, Issy-Les-Moulineaux CI2O I/68 (2006.01) (FR); Kevin Cheeseman, (52) U.S. Cl. Champigny-Sur-Marne (FR); Emmanuel CPC .................................... CI2O I/6886 (2013.01) Conseiller, Paris (FR); Pierre Walrafen, USPC ......................................................... 435/6.11 Montrouge (FR) (57) ABSTRACT (72) Inventors: Aaron BENSIMON, Anthony (FR): Maurizio Ceppi, Issy-Les-Moulineaux Methods for detecting genomic rearrangements in BRCA1 (FR); Kevin Cheeseman, and BRCA2 genes at high resolution using Molecular Comb Champigny-Sur-Marne (FR); Emmanuel ing and for determining a predisposition to a disease or dis Conseiller, Paris (FR); Pierre Walrafen, order associated with these rearrangements including predis Montrouge (FR) position to ovarian cancer or breast cancer. Primers useful for producing probes for this method and kits for practicing the (21) Appl. No.: 13/665,404 methods. Patent Application Publication May 23, 2013 Sheet 1 of 12 US 2013/0130246 A1 85925 379 o : 98. xxxxxx& 3850,75800 :g i s Patent Application Publication May 23, 2013 Sheet 2 of 12 US 2013/0130246 A1 Fig. 1B 845 3.25° 42950 888 O 3690 73800 O700 147600' S. : s 7250, Patent Application Publication May 23, 2013 Sheet 3 of 12 US 2013/0130246 A1 §§§§§§§ gaeae,§§§§** *& ¿(gael ***…) *& Patent Application Publication May 23, 2013 Sheet 4 of 12 US 2013/0130246 A1 s 8x8xx s a ex, N S.& xxx8. wax S. 3. er St. (x : . 8: & s ::::::::::: -: 3. S. 3: NS:8. sgy *i: g.- s: : wa s o & sxxxx Patent Application Publication May 23, 2013 Sheet 5 of 12 US 2013/0130246 A1 ******************************* Patent Application Publication May 23, 2013 Sheet 6 of 12 US 2013/0130246 A1 xxxxxxxxxx $$,žš. wo-wox. -swww.sax888 Patent Application Publication May 23, 2013 Sheet 7 of 12 US 2013/0130246 A1 apoi $$33.3.8 (9eseo)cpººxa?ed |e$$ eiwsiera?avsvozizzzzzzzszez ? Patent Application Publ ication May 23, 2013 Sheet 8 of 12 US 2013/0130246 A1 (zeseo)zxa? $?% 3888-8-8 Patent Application Publication May 23, 2013 Sheet 9 of 12 US 2013/0130246 A1 *· : *****?inxe.------- ***¿. ***?….. *«». *& Patent Application Publicat O May 23, 2013 Sheet 10 of 12 US 2013/0130246 A1 qwzºg-/+z^9?ouo?eolidna! oz-gpxednd |9|8 | 9·61 Patent Application Publication May 23, 2013 Sheet 11 of 12 US 2013/0130246 A1 vae,ºžk? Patent Application Publication May 23, 2013 Sheet 12 of 12 US 2013/0130246 A1 sax £892eup&#38 US 2013/0130246 A1 May 23, 2013 METHODS FOR THE DETECTION, amplification (MLPA) (Casilli et al., 2002), (Hofmann et al., VISUALIZATION AND HIGH RESOLUTION 2002) and high-resolution oligonucleotide array comparative PHYSICAL MAPPING OF GENOMIC genomic hybridization (aCGH) (Rouleau et al., 2007), (Staaf REARRANGEMENTS IN BREAST AND et al., 2008). New approaches that provide both prescreening OVARAN CANCER GENES AND LOC and quantitative information, such as qPCR-HRM and BRCA1 AND BRCA2 USING GENOMIC EMMA, have recently been developed and genomic capture MORSE CODE IN CONJUNCTION WITH combined with massively parallel sequencing has been pro MOLECULAR COMBING posed for simultaneous detection of Small mutations and large rearrangements affecting 21 genes involved in breast CROSS-REFERENCE TO RELATED and ovarian cancer (Walsh et al., 2010). APPLICATIONS 0008 Molecular Combing is a powerful FISH-based tech 0001. The present application claims priority to U.S. Pro nique for direct visualization of single DNA molecules that visional Application No. 61/553,906, filed Oct. 31, 2011, the are attached, uniformly and irreversibly, to specially treated entire contents of which are incorporated herein by reference. glass surfaces (Herrick and Bensimon, 2009): (Schurra and On Oct. 30, 2012, an International Application (PCT/ Bensimon, 2009). This technology considerably improves IBF ; Submission number 1000168920) was also filed the structural and functional analysis of DNA across the with the same title, the entire contents of which are incorpo genome and is capable of visualizing the entire genome at rated herein by reference. high resolution (in the kb range) in a single analysis. Molecu lar Combing is particularly Suited to the detection of genomic BACKGROUND OF THE INVENTION imbalances such as mosaicism, loss of heterozygosity (LOH), copy number variations (CNV), and complex rearrangements 0002 1. Field of the Invention such as translocations and inversions (Caburet et al., 2005), 0003. The invention relates to a method for detecting thus extending the spectrum of mutations potentially detect genomic rearrangements in BRCA1 and BRCA2 genes and able in breast cancer genes. Molecular Combing has been loci at high resolution using Molecular Combing and relates Successfully employed for the detection of large rearrange to a method of determining a predisposition to diseases or ments in BRCA1 (Gadet al., 2001), (Gadet al., 2002a), (Gad disorders associated with these rearrangements including et al., 2003) and BRCA2 (Gad et al., 2002b), using a first predisposition to ovarian cancer or breast cancer. generation “color bar coding screening approach. However, 0004 2. Description of the Related Art these techniques lack resolution and cannot precisely detect 0005 Breast cancer is the most common malignancy in large rearrangements in and around BRCA1 and BRCA2. women, affecting approximately 10% of the female popula 0009. In distinction to the prior art techniques, as disclosed tion. Incidence rates are increasing annually and it is esti herein, the inventors provide a novel Genetic Morse Code mated that about 1.4 million women will be diagnosed with Molecular Combing procedure that provides for high resolu breast cancer annually worldwide and about 460,000 will die tion visual inspection of genomic DNA samples, precise from the disease. Germline mutations in the hereditary breast mapping of mutated exons, precise measurement of mutation and ovarian cancer Susceptibility genes BRCA1 size with robust statistics, simultaneous detection of BRCA1 (MIMH1 13705) and BRCA2 (MIMH600185) are highly pen and BRCA2 genetic structures or rearrangements, detection etrant (King et al., 2003), (Nathanson et al., 2001). Screening of genetic inversions or translocations, and Substantial elimi is important for genetic counseling of individuals with a nation of problems associated with repetitive DNA sequences positive family history and for early diagnosis or prevention such as Alu sequences in BRCA1 and BRCA2 loci. in mutation carriers. When a BRCA1 or BRCA2 mutation is identified, predictive testing is offered to all family members BRIEF SUMMARY OF THE INVENTION older than 18 years. If a woman tests negative, her risk becomes again the risk of the general population. If she tests (0010. The BRCA1 and BRCA2 genes are involved, with positive, a personalized Surveillance protocol is proposed: it high penetrance, in breast and ovarian cancer Susceptibility. includes mammographic screening from an early age, and About 2% to 4% of breast cancer patients with a positive possibly prophylactic Surgery. Chemoprevention of breast family history who are negative for BRCA1 and BRCA2 cancer with anti-estrogens is also currently tested in clinical point mutations can be expected to carry large genomic alter trial and may be prescribed in the future. ations (deletion or duplication) in one of the two genes, and 0006 Most deleterious mutations consist of either small especially BRCA1. However, large rearrangements are frameshifts (insertions or deletions) or point mutations that missed by direct sequencing. Molecular Combing is a pow give rise to premature stop codons, missense mutations in erful FISH-based technique for direct visualization of single conserved domains, or splice-site mutations resulting in aber DNA molecules, allowing the entire genome to be examined rant transcript processing (Szabo et al., 2000). However, at high resolution in a single analysis. A novel predictive mutations also include more complex rearrangements, genetic test based on Molecular Combing is disclosed herein. including deletions and duplications of large genomic regions For that purpose, specific BRCA1 and BRCA2 “Genomic that escape detection by traditional PCR-based mutation Morse Codes” (GMC) were designed, covering coding and screening combined with DNA sequencing (Mazoyer, 2005). non-coding regions and including large genomic portions 0007 Techniques capable of detecting these complex flanking both genes. The GMC is a series of colored signals rearrangements include Southern blot analysis combined distributed along a specific portion of the genomic DNA with long-range PCR or the protein truncation test (PTT), which signals arise from probe hybridization with the probes quantitative multiplex PCR of short fluorescent fragments of the invention. The concept behind the GMC has been (QMPSF) (Hofmannet al., 2002), real-time PCR, fluorescent previously defined in WIPO patent application WO/2008/ DNA microarray assays, multiplex ligation-dependent probe 028931