Patent Application: US-65438203-A

Abstract:
the present invention relates generally to the field of human genetics , and more specifically to the detection of a specific type of germline mutations in the brca1 gene , which will predispose to breast and ovarian cancer . in addition , the invention relates to the molecular genetic mechanism that may have mediated the genesis of these mutations , in particular the role of alu repetitive dna elements present in the intronic regions of brca1 . the invention further relates to somatic mutations of this type in the brca1 gene in human breast and ovarian cancer , and their use in the diagnosis and prognosis of human breast and ovarian cancer . the invention more particularly relates to the screening of this type of brca1 mutations in human genomic dna , which are useful for the diagnosis of inherited predisposition to breast and ovarian cancer .

Description:
the present invention in one of its embodiments , which has been described in detail in the experimental part provides a description and detection in human genomic dna of large genomic deletions in brca1 . in addition , the invention shows involvement of the alu - repeat elements , present at high frequency in the intronic regions of brca1 [ 11 ], in generating a number of these deletions . the invention also contemplates the frequency of these deletions in the dutch population , and their descendance from a common ancestor . we have found that the mutation spectrum of brca1 as resolved up to this point [ 13 , 42 ] has been biased by pcr - based mutation - screening methods such as sscp , the protein truncation test ( ptt ), and direct sequencing , using genomic dna as template . we describe as examples thereof two large genomic deletions , which are not detected by these approaches , and which together comprise 38 % of all brca1 mutations found in a sample of 170 dutch breast cancer families [ 43 , 44 ]. one deletion removes 510 basepairs ( bp ) including exon 22 ( fig1 ) and was found 8 times . the other deletion removes 3835 bp including exon 13 ( fig2 ) and was found 4 times . the haplotypes of the 8 families with the exon 22 deletion were reconstructed by typing 3 intragenic markers ( d17s855 , d17s1322 , d17s1323 ) and 2 flanking markers ( thra1 and d17s1327 ). these haplotypes were completely concordant for the intragenic markers in at least 7 families , and the haplotype conservation extended proximally to thra1 , and distally to d17s1327 , in at least 5 families , to comprise a genetic region of approximately 2 cm . the haplotypes of the 4 families with the exon 13 deletion were reconstructed in a similar way . these haplotypes were completely concordant for the intragenic markers in at least 2 families , and the haplotype conservation extended proximally to thra1 , and distally to d17s1327 , in all 4 families , to comprise a genetic region of approximately 2 cm . molecular characterization of the deletions revealed that the exon 22 deletion starts in intron 21 and ends within the most upstream copy of three head - to - tail arranged alu - elements in intron 22 . a 17 - bp imperfect homology to the intron 22 alu - element was found at the 5 ′ deletion breakpoint ( fig3 ) the 3 ′ breakpoint is closely flanked on either side by two 25 - bp sequences strongly homologous to the alu core - sequence implied to stimulate recombination [ 45 ]. the exon 13 deletion starts in intron 12 in an alu - element ( 112 bp from the 5 ′ end ) and ends in intron 13 in a region which shares very high homology to this element ( fig4 ). both the 5 ′ and the 3 ′ breakpoint are closely flanked on either side by sequences strongly homologous to the 26 - bp alu core - sequence implied to stimulate recombination [ 45 ]. the current invention facilitates the design of pcr - based strategies ( now that the presence of this kind of mutations is known ) to identify the heterozygous presence of the deletions in human genomic dna . oligonucleotide primers can be designed so to immediately flank the deletion breakpoints , and allow the specific amplification of a deletion - junction fragment as a diagnostic endpoint . given the size of the deletions , the wildtype brca1 genomic sequence would remain refractory to pcr - amplification under most standard reaction conditions . pcr - based diagnosis is an essential requirement to scale up throughput in the screening for these mutations . the current invention also pertains to the molecular mechanism which may have generated the genomic deletions in the brca1 gene , especially since this needs to be viewed in a broader sense in that the same kind of phenomenon may be picked up in other genes or in the same gene , but not having anything to do with the inheriting kind of cancer . the current invention thus also pertains to the role of brca1 mutations in non - inherited or sporadic breast cancer . the exon 22 deletion was revealed by southern blot analysis of genomic dna digested with either hindiii or bglii . as probe we used p1424 , which contains ˜ 1 - kb cdna - derived segment from exons 14 - 24 . a carrier of the exon 22 deletion shows aberrant bands of 9 . 3 kb in the hindiii digest and of 6 . 7 kb in the bglii digest . the exon 13 deletion was revealed by southern blot analysis of genomic dna digested with either hindiii or bglii . as probes we used either p11 or p1424 , which contain ˜ 1 - kb cdna - derived segments from exon 11 and exons 14 - 24 , respectively . a carrier of the exon 13 deletion shows an aberrant band of 6 . 4 kb in the hindiii pattern obtained with probe p1424 and of 14 kb in the bglii pattern obtained with probe p11 . to further characterize these deletions , we used intronic amplimers to obtain pcr - products from genomic dna , specifically containing the deletion - junction fragment . amplimers flanking exon 22 generated an aberrant genomic fragment of 1 . 4 kb in dna samples carrying the exon 22 deletion , which turned out to contain a 510 - bp deletion relative to the wildtype sequence ( fig3 ). the deletion affecting exon 22 removes the bases 79505 - 80014 ( 510 bp ) as listed in the genomic sequence of brca1 ( genbank accession nr . l78833 ). as a result , 74 basepairs , corresponding to exon 22 , are missing in the processed mrna - transcript ( bases 79543 - 79615 in genbank accession nr . l78833 ). amplimers flanking exon 13 generated an aberrant genomic fragment of 2 . 7 kb in dna samples carrying the exon 13 deletion , which turned out to contain a 3835 bp deletion relative to the wildtype sequence ( fig4 ). the deletion affecting exon 13 removes the bases 44514 - 48348 ( 3835 basepairs ) as listed in the genomic sequence of brca1 ( genbank accession nr . l78833 ). as a result , 172 basepairs , corresponding to exon 13 , are missing in the processed mrna - transcript ( nucleotides 46156 - 46327 in genbank accession nr . l78833 ). we examined 142 breast cancer families in which thusfar no brca1 or brca2 mutation had been found ( refs . 43 , 44 and our unpublished results ) for the presence of the exon 13 and exon 22 deletions . they were found in 4 and 8 families , respectively . together with previous mutation screening results , using ptt and direct sequencing [ 44 ], these deletions thus comprise 12 / 32 ( 38 %) of all families in which a brca1 mutation has been detected to date . three intragenic and 2 flanking markers were used to reconstruct the disease haplotype for each of the research families carrying either the 510 - bp or 3 . 8 - kb deletion . strong conservation of allele - lengths was observed at the intragenic loci among the haplotypes carrying the same deletion , in agreement with their descent from a common ancestor . the haplotype in the dutch population that carries the 510 - bp deletion around exon 22 is characterized by a 155 - bp allele at the microsatellite marker d17s855 in intron 20 , a 122 - bp allele at microsatellite marker d17s1322 in intron 19 , and a 151 - bp allele at microsatellite marker d17s 1323 in intron 12 . the haplotype in the dutch population that carries the 3835 - bp deletion around exon 13 is characterized by a 151 - bp allele at d17s855 , a 122 - bp allele at d17s1322 , and a 151 - bp allele at d17s1323 in intron 12 . the primer sequences used to detect these alleles are : for d17s 1322 : forward ( f ) 5 ′ ctagcctgggcaacaaacga 3 ′ ( seq . id . no . : 1 and reverse ( r ) 5 ′ gcaggaagcaggaatggaac 3 ′ ( seq . id . no . : 2 ); for d17s855 : f 5 ′ ggatggccttttagaaagtgg 3 ′ ( seq . id . no . : 3 ) and r 5 ′ acacagacttgtcctactgc 3 ′ ( seq . id . no . : 4 ); for d17s1323 : f 5 ′ taggagatggattattggtg 3 ′ ( seq . id . no . : 5 ) and r 5 ′ aagcaactttgcaatgagtg 3 ′ ( seq . id . no . : 6 ). pcr conditions have been described elsewhere [ 44 ]. isolation of genomic dna and total rna from freshly taken blood samples , and preparation of first - strand cdna by reverse transcription , has been described [ 43 ]. exons 12 - 24 were amplified from first - strand cdna products obtained by reverse transcription using the following primers for the first pcr : f 5 ′ tcacagtgcagtgaattggaag 3 ′ ( seq . id . no . : 7 ) and r 5 ′ gtagccaggacagtagaaggactg 3 ′ ( seq . id . no . : 8 ). the obtained pcr - products were used as template for a second pcr of exons 12 - 24 using nested primers ( f 5 ′ gaagaaagaggaacgggcttgg 3 ′ ( seq . id . no . : 9 ) and r 5 ′ ggccactttgtaagctcattc 3 ′ ( seq . id . no . : 10 )). pcr conditions were as described previously [ 43 ]. five μl of the final pcr products are analysed on a 1 % agarose gel . exons 12 - 24 were amplified from first - strand cdna products obtained by reverse transcription using the following primers for the first pcr : f 5 ′ tcacagtgcagtgaattggaag 3 ′ ( seq . id . no . : 7 ) and r 5 ′ gtagccaggacagtagaaggactg 3 ′ ( seq . id . no . : 8 ). the obtained pcr - products were used as template for a second pcr of exons 20 - 24 using nested primers ( f 5 ′ aaccaccaaggtccaaagc 3 ′ ( seq . id . no . : 11 ) and r 5 ′ gtagccaggacagtagaaggactg 3 ′ ( seq . id . no . : 12 )). pcr conditions were as described previously [ 43 ]. five μl of the final pcr products are analysed on a 1 % agarose gel . genomic pcr of the 3835 - bp deletion spanning exon 13 . a pcr reaction of 50 μl contains 200 ng of genomic dna , 10 pmol primers ( f 5 ′ taggagatggattattggtg 3 ′ ( seq . id . no . : 5 ) and r 5 ′ tacgtgggttcaactgaagc 3 ′ ( seq . id . no . : 13 )), 0 . 75 units amplitaq taq polymerase ( perkin - elmer - cetus ), and 5 μl of 10 × itp / bsa buffer ( 500 mm kcl 100 mm tris - hcl ph 8 . 4 , 25 mm mgcl 2 , 2 mg / ml bsa , 2 mm dntps ). this mixture is heated at 94 ° c . for 5 minutes , followed by 35 cycles of pcr ( at 94 ° c . for 45 seconds , at 52 ° c . for 1 min . and at 72 ° c . for 2 . 5 mm on a perkin - elmer - cetus dna thermal cycler ). the pcr is concluded by an incubation at 72 ° c . for 6 minutes . five μl of the pcr products are analysed on a 1 % agarose gel . genomic pcr of the 510 - bp deletion spanning exon 22 . a pcr reaction of 50 μl contains 300 ng of genomic dna , 10 pmol primers ( f 5 ′ tcccattgagaggtcttgct 3 ′ ( seq . id . no . : 14 ) and r 5 ′ actgtgctactcaagcacca 3 ′ ( seq . id . no . : 15 )), 0 . 75 u amplitaq taq polymerase ( perkin - elmer - cetus ), 5 μl optiprime buffer # 6 ( stratagene ) and 0 . 1 mm dntps . thermal cycles are as described for the deletion of 3 . 8 kb . five μl of the pcr products are analysed on a 1 . 5 % agarose gel . five μg of genomic dna is digested with either the restriction endonuclease bglii or hindiii . agarose gels ( 0 . 8 %) are run at 30v for 16 hr in tae buffer ( 40 mm tris - hac ph 8 . 3 , 1 mm edta ). procedures for denaturing , and transferring the separated dna to nylon membranes ( hybond n +, amersham ) have been described [ 46 ]. as probes we used pcr - products obtained from a clone containing the complete brca1 - cdna , and purified by using the qiaquick pcr purification kit from qiagen . probe - 11 ( p11 ) derives entirely from exon 11 and was obtained with the primers f 5 ′ gaaaaaaaagtacaaccaaatgcc 3 ′ ( seq . id . no . : 16 ) and r 5 ′ agcccacttcattagtactggaac 3 ′ ( seq . id . no . : 17 ), and probe - 1424 ( p1424 ) contains exons 14 - 24 and was obtained with the primers f 5 ′ taccctataagccagaatccagaa 3 ′ ( seq . id . no . : 18 ); and r 5 ′ ggccactttgtaagctcattc 3 ′ ( seq . id . no . : 19 ). purified fragments were labelled using the megaprime dna labelling system from amersham according to suppliers protocols . hybridizations were carried out at 65 ° c . in 125 mm na2hpo4 . 2h2o , 7 % sds , 10 % peg - 6000 , 1 mm edta . final washing was in 45 mm nacl , 4 . 5 mm na - citrate ph 7 . 0 , 0 . 1 % sds , at 65 ° c . for 30 minutes . fig1 . schematic representation of the genomic deletion spanning exon 22 of brca1 . the intronic regions are drawn to scale relative to one another , and the exonic region are drawn to scale relative to one another , but not to intronic regions . the positions of the restriction endonucleases hindiii and bglii , used in southern blot analysis , are indicated . the arrows indicate the presence and orientation of an alu - element . fig2 . sequence of exon 22 ( uppercase ) and its flanking intron - sequence ( lower case ) ( seq . id . no . : 20 ). the numbers refer to the genomic sequence of brca1 ( genbank accession nr . l78833 ). starting and ending positions of the 510 - bp deletion are indicated by hooked arrows and affect positions 79505 - 80014 . the first 241 bp of an alu - element are depicted in italics , and the boxed sequences are imperfect copies ( 1 and 5 mismatches , respectively ) of a common 26 - bp core sequence involved in recombinations leading to gene rearrangements in the ldlr gene [ 45 ]. a stretch of 17 bp at the 5 ′ junction of the deletion is homologous to a 19 - bp stretch 37 bp upstream of the 3 ′ deletion - breakpoint ( underlined with arrows ). fig3 . schematic representation of the genomic deletion spanning exon 13 of brca1 . the intronic regions are drawn to scale relative to one another , and the exonic region are drawn to scale relative to one another , but not to intronic regions . the positions of the restriction endonucleases hindiii and bglii , used in southern blot analysis , are indicated . the arrowheads indicate the presence and orientation of an alu - element . fig4 . aligned sequences of intronic regions flanking exon 13 ( seq . id . nos . : 21 and 22 ), and of the deletion - junction fragment ( jnctn ) ( seq . id . no . : 23 ). the upper sequence of each alignment corresponds to intron 12 sequences ( seq . id . no . : 21 ), the lower sequence intron 13 sequences ( seq . id . no . : 22 ). the numbers refer to the genomic sequence of brca1 ( genbank accession nr . l78833 ). the boxed sequence indicates the 10 bp where the recombination took place that led to the deletion of 3835 bp . the intron 12 sequence depicted here represents the first 180 bp of an alu - element . the intron 12 region 44481 - 44551 shares an 85 % identity with the intron 13 region 48316 - 48386 . the underlined sequences are imperfect copies of a common 26 - bp core sequence involved in recombinations leading to gene rearrangements in the ldlr gene [ 45 ]. 1 . slattery m l , and kerber r a ( 1993 ) a comprehensive evaluation of family history and breast cancer risk : the utah population database . jama 270 : 1563 - 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