Converting diploidy to haploidy for genetic diagnosis

Abstract

Detection of mutations associated with hereditary diseases is complicated by the diploid nature of mammalian cells. Mutations present in one allele are often masked by the wild-type sequence of the other allele. Individual alleles can be isolated from every chromosome within somatic cell hybrids generated from a single fusion. Nucleic acids from the hybrids can be analyzed for mutations in an unambiguous manner. This approach was used to detect two cancer-causing mutations that had previously defied genetic diagnosis. One of the families studied, Warthin Family G, was the first kindred with a hereditary colon cancer syndrome described in the biomedical literature.

1 . A cell line comprising mouse embryonic fibroblasts which are transformed by oncogenes, wherein said fibroblasts are defective in a mismatch repair gene which causes microsatellite instability.  
   
   
       2 . The cell line of  claim 1  wherein the fibroblasts are deficient in hypoxanthine phosphoribosyl transferase (HPRT).  
   
   
       3 . The cell line of  claim 1  wherein said oncogenes comprise Ras.  
   
   
       4 . The cell line of  claim 1  wherein said oncogenes comprise E1A.  
   
   
       5 . The cell line of  claim 1  wherein said oncogenes comprise Ras and E1A.  
   
   
       6 . A method of making a rodent cell recipient for fusion with mammalian cells comprising: 
 transforming rodent cell embryonic fibrtoblasts with oncogenes, wherein said fibroblasts are defective in a mismatch repair gene which causes microsatellite instability; and    selecting HPRT-deficient cells by growing in 2-amino-6-mercaptopurine.    
   
   
       7 . The cell line of  claim 6  wherein said oncogenes comprise oncogene E1A.  
   
   
       8 . The cell line of  claim 6  wherein said oncogenes comprise oncogene Ras.  
   
   
       9 . The cell line of  claim 6  wherein said oncogenes comprise oncogene E1A and Ras.  
   
   
       10 . The method of  claim 6  wherein the step of detecting comprises identifying a set comprising a plurality of distinctive markers on the second and third non-rodent mammalian chromosomes.  
   
   
       11 . The method of  claim 10  wherein the distinctive markers comprise microsatellite markers.  
   
   
       12 . The method of  claim 10  wherein the distinctive markers comprise polymorphic markers.  
   
   
       13 . A method of identifying a haplotype of a non-rodent mammalian chromosome comprising: 
 fusing cells of a non-rodent mammal to rodent cell recipients to form non-rodent mammal-rodent cell hybrids;    selecting for fused cell hybrids by selecting for a first marker contained on a rodent chromosome and for a second marker contained on a first non-rodent mammalian chromosome, to form a population of fused cell hybrids;    detecting among the population of fused cell hybrids a subset of hybrids which are haploid for a second non-rodent mammalian chromosome which is not the same chromosome as the first non-rodent mammalian chromosome and which was not selected;    analyzing said subset of hybrids to detect a plurality of distinctive markers on the second non-rodent mammalian chromosome.    
   
   
       14 . The method of  claim 13  wherein the marker set comprises polymorphic markers.  
   
   
       15 . The method of  claim 13  wherein the marker set comprises microsatellite markers.  
   
   
       16 . A method of correlating a polymorphic marker with expression or reduced expression of a gene in a non-rodent individual, comprising the steps of: 
 (a) fusing cells of the non-rodent individual to rodent cell recipients to form non-rodent/rodent cell hybrids;    (b) selecting for fused cell hybrids by selecting for a first selectable marker contained on a rodent chromosome and for a second selectable marker contained on a first non-rodent individual chromosome, to form a population of fused cell hybrids;    (c) detecting among the population of fused cell hybrids a subset of hybrids which are haploid for a second non-rodent individual chromosome which is not the same chromosome as the first non-rodent individual chromosome and which was not selected;    (d) analyzing said subset of hybrids to detect a polymorphic marker on the second non-rodent individual chromosome;    (e) assaying for expression of a gene on the second non-rodent individual chromosome; and    (f) identifying the polymorphic marker as correlated with expression of the gene if the subset of hybrids comprises the polymorphic marker and the gene is expressed in the hybrids or identifying the polymorphic marker as correlated with reduced expression of the gene if the subset of hybrids comprises the polymorphic marker and expression of the gene is reduced in the hybrids.    
   
   
       17 . The method of  claim 16  wherein the polymorphic marker is a single nucleotide polymorphism.  
   
   
       18 . The method of  claim 16  wherein the polymorphic marker is a microsatellite marker.  
   
   
       19 . The method of  claim 16  wherein the polymorphic marker is a plurality of polymorphic markers on the second non-rodent individual chromosome.  
   
   
       20 . The method of  claim 16  wherein the polymorphic marker is a mutation.  
   
   
       21 . The method of  claim 16  wherein an mRNA product of the gene is analyzed in the subset of hybrids.  
   
   
       22 . The method of  claim 16  wherein a protein product of the gene is analyzed in the subset of hybrids.  
   
   
       23 . The method of  claim 16  wherein the gene is analyzed in the subset of hybrids.  
   
   
       24 . The method of  claim 16  wherein the non-rodent individual is a human.  
   
   
       25 . The method of  claim 16  wherein the subset of hybrids is analyzed to detect a plurality of polymorphic markers.  
   
   
       26 . The method of  claim 16  wherein the subset of hybrids is analyzed to detect polymorphic markers in at least two different genes.  
   
   
       27 . A method of using a correlation between a polymorphic marker and expression or reduced expression of a gene to select a medical intervention for a non-rodent individual, comprising the steps of: 
 (a) assaying a biological sample obtained from the non-rodent individual for a polymorphism which is correlated with expression of a gene, wherein the correlation has been determined by a method comprising the steps of: 
 (1) fusing cells of the non-rodent individual to rodent cell recipients to form non-rodent/rodent cell hybrids;  
 (2) selecting for fused cell hybrids by selecting for a first selectable marker contained on a rodent chromosome and for a second selectable marker contained on a first non-rodent individual chromosome, to form a population of fused cell hybrids;  
 (3) detecting among the population of fused cell hybrids a subset of hybrids which are haploid for a second non-rodent individual chromosome which is not the same chromosome as the first non-rodent individual chromosome and which was not selected;  
 (4) analyzing said subset of hybrids to detect a polymorphic marker on the second non-rodent individual chromosome;  
 (5) assaying for expression of a gene on the second non-rodent individual chromosome; and  
 (6) identifying the polymorphic marker as correlated with expression of the gene if the subset of hybrids comprises the polymorphic marker and the gene is expressed in the hybrids or identifying the polymorphic marker as correlated with reduced expression of the gene if the subset of hybrids comprises the polymorphic marker and expression of the gene is reduced in the hybrids; and  
   (b) selecting a medical intervention based on the presence or absence of the polymorphic marker in the biological sample.    
   
   
       28 . The method of  claim 27  wherein the medical intervention is a prophylactic intervention.  
   
   
       29 . The method of  claim 27  wherein the polymorphic marker predisposes the non-rodent individual to a disorder.  
   
   
       30 . The method of  claim 27  wherein the polymorphic marker is causally related to a disorder.  
   
   
       31 . The method of  claim 27  wherein the polymorphic marker is associated with responsiveness to a drug and wherein the medical intervention is administration of the drug.  
   
   
       32 . The method of  claim 27  wherein the polymorphic marker is associated with resistance to a first drug useful for treating the disorder and wherein the medical intervention is administration of a second drug useful for treating the disorder.