Mono-allelic mutation analysis for identifying germline mutations

Abstract

A diagnostic strategy for detection of inherited diseases caused by germline mutations is based on somatic cell hybridization. Each allele of a human gene involved in the inherited disease is isolated in a somatic cell hybrid. The products of the isolated human allele are then observed in the absence of the other allele of the human.

We claim:  
     
       1. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
       obtaining cells of the human;  
       fusing said cells to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome, to form human-rodent cell hybrids;  
       testing said human-rodent cell hybrids to confirm the presence of said chromosome of the human in said hybrid;  
       testing said hybrids which contains  contain said chromosome to detect a protein product of said first gene, absence of said protein product or diminished amounts of said protein product indicating the presence of a mutation in the first gene of the human.  
     
     
       2. The method of  claim 1  wherein the first gene is APC. 
     
     
       3. The method of  claim 1  wherein the first gene is hMSH2. 
     
     
       4. The method of  claim 1  wherein the first gene is p53. 
     
     
       5. The method of  claim 1  wherein the rodent cell recipients are auxotrophic mutants. 
     
     
       6. The method of  claim 1  wherein the rodent cell recipients are biosynthetic mutants. 
     
     
       7. The method of  claim 1  wherein the cells of the human are peripheral blood lymphocytes. 
     
     
       8. The method of  claim 1  wherein the fusion is performed in the presence of polyethylene glycol. 
     
     
       9. The method of  claim 1  wherein the fusion is electrophoretic. 
     
     
       10. The method of  claim 1  wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene. 
     
     
       11. The method of  claim 1  wherein said step of testing to detect a protein product is performed by Western blotting. 
     
     
       12. The method of  claim 1  wherein said step of testing to detect a protein product is performed by an enzyme reaction. 
     
     
       13. The method of  claim 1  wherein said step of testing to detect a protein product is performed by assaying for function of the protein product. 
     
     
       14. The method of  claim 11  wherein an antibody which is specifically immunoreactive with the protein product of the first gene is used in the Western blotting, wherein said antibody is not immunoreactive with a homologous protein product of the rodent cell. 
     
     
       15. The method of  claim 1  further comprising the step of: 
       testing a human-rodent cell hybrid which contains a wild-type allele of the first gene, to confirm that said allele can be expressed in human-rodent cell hybrids.  
     
     
       16. The method of  claim 13  wherein one or more rodent protein product homologues of the human protein product of the gene of interest are inactivated by mutation. 
     
     
       17. The method of  claim 1  further comprising the step of: 
       if a mutation in the first gene of the human is indicated, determining a haplotype for the chromosome of the human; and  
       testing one or more family members for the presence of said haplotype, wherein the presence of said haplotype implies the presence of the mutation.  
     
     
       18. The method of  claim 17  wherein the haplotype comprises an allele of each of at least two microsatellite markers flanking said first gene. 
     
     
       19. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
       obtaining peripheral blood lymphocytes of the human; fusing said peripheral blood lymphocytes to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome, to form human-rodent cell hybrids;  
       testing said human-rodent cell hybrids to confirm the presence of said chromosome of the human in said hybrids, wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene;  
       testing said hybrids which contains  contain said chromosome to detect a protein product of said first gene by Western blotting, absence of said protein product or diminished amounts of said protein product indicating the presence of a mutation in the first gene of the human.  
     
     
       20. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
         fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome, to form human - rodent cell hybrids;    
         testing said human - rodent cell hybrids to confirm the presence of said chromosome of the human in said hybrid;    
       
         testing said hybrids which contain said chromosome to detect a protein product of said first gene, absence of said protein product or diminished amounts of said protein product indicating the presence of a mutation in the first gene of the human. 
       
     
     
       21. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
       ( a )  fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome, to form human - rodent cell hybrids;    
       ( b )  testing said human - rodent cell hybrids to confirm the presence of said chromosome of the human in said hybrid;    
       ( c )  testing said hybrids formed in step  ( a )  which contain said chromosome to detect a mutation in the first gene of the human.   
     
     
       22. The method of  claim 21  wherein the step of testing to detect a mutation employs a polymerase chain reaction ( PCR ). 
     
     
       23. The method of  claim 21  wherein the step of testing to detect a mutation employs automated sequencing. 
     
     
       24. The method of  claim 21  wherein the step of testing to detect a mutation employs a reverse transcription- polymerase chain reaction  ( RT - PCR ). 
     
     
       25. The method of  claim 21  wherein the step of testing to detect a mutation subjects DNA of said hybrids to mutation analysis. 
     
     
       26. The method of  claim 20  wherein the first gene is APC. 
     
     
       27. The method of  claim 20  wherein the first gene is hMSH 2 . 
     
     
       28. The method of  claim 20  wherein the first gene is p 53 . 
     
     
       29. The method of  claim 20  wherein the rodent cell recipients are auxotrophic mutants. 
     
     
       30. The method of  claim 20  wherein the rodent cell recipients are biosynthetic mutants. 
     
     
       31. The method of  claim 20  wherein the cells of the human are peripheral blood lymphocytes. 
     
     
       32. The method of  claim 20  wherein the fusion is performed in the presence of polyethylene glycol. 
     
     
       33. The method of  claim 20  wherein the fusion is electrophoretic. 
     
     
       34. The method of  claim 20  wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene. 
     
     
       35. The method of  claim 20  further comprising the step of: 
       
         if a mutation in the first gene of the human is indicated, determining a haplotype for the chromosome of the human; and  
       
       
         testing one or more family members for the presence of said haplotype, wherein the presence of said haplotype implies the presence of the mutation. 
       
     
     
       36. The method of  claim 35  wherein the haplotype comprises an allele of each of at least two microsatellite markers flanking said first gene. 
     
     
       37. The method of  claim 21  wherein the first gene is APC. 
     
     
       38. The method of  claim 21  wherein the first gene is hMSH 2 . 
     
     
       39. The method of  claim 21  wherein the first gene is p 53 . 
     
     
       40. The method of  claim 21  wherein the rodent cell recipients are auxotrophic mutants. 
     
     
       41. The method of  claim 21  wherein the rodent cell recipients are biosynthetic mutants. 
     
     
       42. The method of  claim 21  wherein the cells of the human are peripheral blood lymphocytes. 
     
     
       43. The method of  claim 21  wherein the fusion is performed in the presence of polyethylene glycol. 
     
     
       44. The method of  claim 21  wherein the fusion is electrophoretic. 
     
     
       45. The method of  claim 21  wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene. 
     
     
       46. The method of  claim 21  further comprising the steps of: 
       
         if a mutation in the first gene of the human is indicated, determining a haplotype for the chromosome of the human; and  
       
       
         testing one or more family members for the presence of said haplotype, wherein the presence of said haplotype implies the presence of the mutation. 
       
     
     
       47. The method of  claim 46  wherein the haplotype comprises an allele of each of at least two microsatellite markers flanking said first gene. 
     
     
       48. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
         fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome, to form human - rodent cell hybrids;    
         testing said human - rodent cell hybrids to confirm the presence of said chromosome of the human in said hybrid;    
       
         testing said hybrids which contain said chromosome to detect a haplotype, wherein the haplotype has been associated with a mutation in the first gene. 
       
     
     
       49. The method of  claim 20  wherein said step of testing to detect a protein product is performed by Western blotting. 
     
     
       50. The method of  claim 20  wherein said step of testing to detect a protein product is performed by an enzyme reaction. 
     
     
       51. The method of  claim 20  wherein said step of testing to detect a protein product is performed by assaying for function of the protein product. 
     
     
       52. The method of  claim 49  wherein an antibody which is specifically immunoreactive with the protein product of the first gene is used in the Western blotting, wherein said antibody is not immunoreactive with a homologous protein product of the rodent cell. 
     
     
       53. The method of  claim 20  further comprising the step of: 
         testing a human - rodent cell hybrid which contain a wild - type allele of the first gene, to confirm that said allele can be expressed in human - rodent cell hybrids.   
     
     
       54. The method of  claim 51  wherein one or more rodent protein product homologues of the human protein product of the gene of interest are inactivated by mutation. 
     
     
       55. The method of  claim 20  wherein the presence of said chromosome is confirmed by detecting a linked polymorphic marker. 
     
     
       56. The method of  claim 21  wherein the presence of said chromosome is confirmed by detecting a linked polymorphic marker. 
     
     
       57. The method of  claim 48  wherein the presence of said chromosome is confirmed by detecting a linked polymorphic marker. 
     
     
       58. The method of  claim 21  wherein the mutation results in reduced expression of the full- length gene product.   
     
     
       59. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
         testing human - rodent cell hybrids formed by:    
       
         fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome,  
       
       
         to confirm the presence of said chromosome of the human in said hybrid;  
       
         testing said human - rodent cell hybrids which contain said chromosome to detect a protein product of said first gene, absence of said protein product or diminished amounts of said protein product indicating the presence of a mutation in the first gene of the human.   
     
     
       60. The method of  claim 59  wherein the first gene is APC. 
     
     
       61. The method of  claim 59  wherein the first gene is hMSH 2 . 
     
     
       62. The method of  claim 59  wherein the first gene is p 53 . 
     
     
       63. The method of  claim 59  wherein the rodent cell recipients are auxothropic mutants. 
     
     
       64. The method of  claim 59  wherein the rodent cell recipients are biosynthetic mutants. 
     
     
       65. The method of  claim 59  wherein the cells of the human are peripheral blood lymphocytes. 
     
     
       66. The method of  claim 59  wherein the fusion was performed in the presence of polyethylene glycol. 
     
     
       67. The method of  claim 59  wherein the fusion was electrophoretic. 
     
     
       68. The method of  claim 59  wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene. 
     
     
       69. The method of  claim 59  wherein said step of testing to detect a protein product is performed by Western blotting. 
     
     
       70. The method of  claim 59  wherein said step of testing to detect a protein product is performed by an enzyme reaction. 
     
     
       71. The method of  claim 59  wherein said step of testing to detect a protein product is performed by assaying for function of the protein product. 
     
     
       72. The method of  claim 69  wherein an antibody which is specifically immunoreactive with the protein product of the first gene is used in the Western blotting, wherein said antibody is not immunoreactive with a homologous protein product of the rodent cell. 
     
     
       73. The method of  claim 59  further comprising the step of: 
         testing a human - rodent cell hybrid which contains a wild - type allele of the first gene, to confirm that said allele can be expressed in human - rodent cell hybrids.   
     
     
       74. The method of  claim 71  wherein one or more rodent protein product homologues of the human protein product of the gene of interest are inactivated by mutation. 
     
     
       75. The method of  claim 59  further comprising the step of: 
       
         if a mutation in the first gene of the human is indicated, determining a haplotype for the chromosome of the human; and  
       
       
         testing one or more family members for the presence of said haplotype, wherein the presence of said haplotype implies the presence of the mutation. 
       
     
     
       76. The method of  claim 75  wherein the haplotype comprises an allele of each of at least two microsatellite markers flanking said first gene. 
     
     
       77. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
         testing human - rodent cell hybrids formed by:    
       
         fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome,  
       
       
         to confirm the presence of said chromosome of the human in said hybrid, wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene;  
       
         testing said human - rodent cell hybrids which contain said chromosome to detect a protein product of said first gene by Western blotting, absence of said protein product or diminished amounts of said protein product indicating the presence of a mutation in the first gene of the human.   
     
     
       78. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
       ( a )  testing human - rodent cell hybrids formed by:    
       
         fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome,  
       
       
         to confirm the presence of said chromosome of the human in said hybrid;  
       
       ( b )  testing said hybrids formed in step  ( a )  which contain said chromosome to detect a mutation in the first gene of the human.   
     
     
       79. The method of  claim 78  wherein the step of testing to detect a mutation employs a polymerase chain reaction ( PCR ). 
     
     
       80. The method of  claim 78  wherein the step of testing to detect a mutation employs automated sequencing. 
     
     
       81. The method of  claim 78  wherein the step of testing to detect a mutation employs a reverse transcription- polymerase chain reaction  ( RT - PCR ). 
     
     
       82. The method of  claim 78  wherein the step of testing to detect a mutation subjects DNA of said hybrids to mutation analysis. 
     
     
       83. The method of  claim 78  wherein the first gene is APC. 
     
     
       84. The method of  claim 78  wherein the first gene is hMSH 2 . 
     
     
       85. The method of  claim 78  wherein the first gene is p 53 . 
     
     
       86. The method of  claim 78  wherein the rodent cell recipients are auxotrophic mutants. 
     
     
       87. The method of  claim 78  wherein the rodent cell recipients are biosynthetic mutants. 
     
     
       88. The method of  claim 78  wherein the cells of the human are peripheral blood lymphocytes. 
     
     
       89. The method of  claim 78  wherein the fusion was performed in the presence of polyethylene glycol. 
     
     
       90. The method of  claim 78  wherein the fusion was electrophoretic. 
     
     
       91. The method of  claim 78  wherein the presence of said chromosome is tested by identifying the presence of microsatellite markers flanking the first gene. 
     
     
       92. The method of  claim 78  further comprising the step of: 
       
         if a mutation in the first gene of the human is indicated, determining a haplotype for the chromosome of the human; and  
       
       
         testing one or more family members for the presence of said haplotype, wherein the presence of said haplotype implies the presence of the mutation. 
       
     
     
       93. The method of  claim 92  wherein the haplotype comprises an allele of each of at least two microsatellite markers flanking said first gene. 
     
     
       94. A method of detecting mutations in a first gene of a human, wherein the first gene has been previously mapped to a human chromosome, comprising the steps of: 
         testing human - rodent cell hybrids formed by:    
       
         fusing cells of the human to rodent cell recipients which carry a mutation which can be complemented by a second gene which has been mapped to said chromosome,  
       
       
         to confirm the presence of said chromosome of the human in said hybrid;  
       
       
         testing said hybrids which contain said chromosome to detect a haplotype, wherein the haplotype has been associated with a mutation in the first gene. 
       
     
     
       95. The method of  claim 78  wherein the presence of said chromosome is confirmed by detecting a linked polymorphic marker. 
     
     
       96. The method of  claim 94  wherein the presence of said chromosome is confirmed by detecting a linked polymorphic marker. 
     
     
       97. The method of  claim 78  wherein the mutation results in reduced expression of the full- length gene product.