Methods for Identifying Genomic Deletions

Abstract

The genomic locus responsible for Van Buchem's disease is narrowed to an approximately 92 kb region of human chromosome 17 at 17q21. Individuals afflicted with or carriers of Van Buchem's disease exhibit a 52 kb deletion within this 92 kb region. Methods are provided that permit the differentiation between individuals homozygous for and therefore afflicted with Van Buchem's disease, individuals heterozygous for and therefore carriers of Van Buchem's disease, and individuals who are normal with respect to Van Buchem's disease. Also provided are general methodologies for the detection of a wide variety of large genomic deletions.

1 . A method for distinguishing between an individual who is homozygous for a large genomic deletion, an individual who is heterozygous for a large genomic deletion and an individual who is negative for a large genomic deletion, said method comprising:
 (a) obtaining a sample of genomic DNA from said individual;   (b) performing a first amplification reaction with a first oligonucleotide primer pair comprising a first oligonucleotide and a second oligonucleotide wherein said first oligonucleotide is complementary to a nucleotide sequence upstream of said genomic deletion and said second oligonucleotide is complementary to a nucleotide sequence downstream of said genomic deletion;   (c) performing a second amplification reaction with a second oligonucleotide primer pair comprising third oligonucleotide and a fourth oligonucleotide wherein said third oligonucleotide is complementary to a nucleotide sequence either the upstream or downstream of said genomic deletion and said fourth oligonucleotide is complementary to a nucleotide sequence within said genomic deletion; and   (d) detecting the product of the amplification reactions of (b)and (c); wherein a positive amplification reaction of (b) and a negative amplification reaction of (c) indicates an individual that is homozygous for said large genomic deletion; a positive amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual that is heterozygous for said large genomic deletion; and a negative amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual that is negative for said large genomic deletion.   
     
     
         2 . The method of  claim 1 , wherein said large genomic deletion comprises a deletion selected from the group consisting of:
 (a) deletions of at least 2 kilobases;   (b) deletions of at least 5 kilobases;   (c) deletions of at least 10 kilobases;   (d) deletions of at least 25 kilobases; and   (e) deletions of at least 50 kilobases.   
     
     
         3 . The method of  claim 1  wherein said amplification reaction comprises the polymerase chain reaction. 
     
     
         4 . The method of  claim 1  wherein the presence of said large genomic deletion indicates an individual who is either a carrier of or afflicted with a genetic disease. 
     
     
         5 . The method of  claim 4  wherein said genetic disease is Van Buchem's disease. 
     
     
         6 . A method for identifying a carrier of Van Buchem's disease, said method comprising:
 (a) obtaining a sample of genomic DNA from said individual;   (b) performing a first polymerase chain reaction with a first oligonucleotide primer pair comprising a first oligonucleotide and a second oligonucleotide wherein said first oligonucleotide is complementary to a nucleotide sequence upstream of the 51,719 bp sequence depicted in SEQ ID NO:2 and said second oligonucleotide is complementary to a nucleotide sequence downstream of the 51,719bp sequence depicted in SEQ ID NO:2;   (c) performing a second polymerase chain reaction with a second oligonucleotide primer pair comprising a third oligonucleotide and a fourth oligonucleotide wherein said third oligonucleotide is complementary to either the nuceotide sequence upstream or downstream of said 51,719 bp sequence depicted in SEQ ID NO:2 and said fourth oligonucleotide is complementary to a nucleotide sequence within the 51,719 bp sequence depicted in SEQ 10 NO:2; and   (d) detecting the product of the polymerase chain reactions of (b)and (c), wherein a positive polymerase chain reaction of (b) and a negative polymerase chain reaction of (c) indicates an individual afflicted with Van Buchem's disease; a positive polymerase chain reaction of (b) and a positive polymerase chain reaction of (c) indicates an individual that is a carrier of Van Buchem's disease; and a negative polymerase chain reaction of (b) and a positive polymerase chain reaction of (c) indicates an individual that is neither afflicted with nor a carrier of Van Buchem's disease.   
     
     
         7 . The method of  claim 6  wherein said first oligonucleotide pair is selected from the group consisting of I2952/VBspan1 (SEQ ID NO:84/SEQ ID NO:85), SpanIF/SpanIR (SEQ ID NO:86/SEQ ID NO:87), Span2F/Span2R (SEQ ID NO:88/SEQ ID NO:89), Wt2F/VBspan1 (SEQ ID NO:91/SEQ ID NO:85) and VBspan2/VBspan1 (SEQ ID NO: I04/SEQ ID NO: 85). 
     
     
         8 - 11 . (canceled) 
     
     
         12 . A method for detecting an individual who is afflicted with or a carrier for Van Buchem's disease, said method comprising detecting the nucleotide sequence spanning the deletion breakpoint as depicted by  FIG. 2 , the sequence of which is provided in SEQ ID NO: 101, wherein said nucleotide sequence spanning said deletion breakpoint comprises a nucleotide sequence selected from the group consisting of 5′-ACCATGCCCGGCTAAT-3′, 5′-CTACCATGCCCGGCTAATTTT-3′ and 5′-TGGGATTACAGGTGCATGCTACCATGCCCGGCTAATTTTTTTGTATTTTTTTA-3′, and wherein the presence of a deletion breakpoint indicates an individual who is either afflicted with or a carrier for Van Buchem's disease. 
     
     
         13 - 31 . (canceled)