Methods of identifying point mutations in a genome that affect the risk for or the age of appearance of disease

Abstract

The invention relates to a method for identifying inherited point mutations in a targeted region of the genome in a large population of individuals and determining which inherited point mutations are deleterious, harmful or beneficial. Deleterious mutations are identified directly by a method of recognition using the set of point mutations observed in a large population of juveniles. Harmful mutations are identified by comparison of the set of point mutation observed in a large set of juveniles and a large set of aged individuals of the same population. Beneficial mutations are similarly identified.

1 . A method for identifying genes that carry one or more alleles that affect the risk for or age of appearance of a disease, comprising:
 a) determining the number of all detectable point mutations in genes or gene segments of a control population not afflicted with said disease, and calculating the sum of the frequencies of all point mutations identified in each gene or segment thereof;   b) determining the number of all detectable point mutations in genes or gene segments of a case population afflicted with the disease, and calculating the sum of the frequencies of all point mutations identified in each gene or segment thereof; and   c) comparing the sum of the frequencies of point mutations associated with a particular gene or gene segment in the unafflicted population calculated in a) with the sum of the frequencies of point mutations that are found gene or gene segment in the case population calculated in b); wherein a significant difference in the sum of the frequencies of point mutations in the case population indicates that the gene or gene segment affects the risk for or age of appearance of the disease.   
     
     
         2 . The method of  claim 1 , wherein said disease is a mortal disease or a mental disease. 
     
     
         3 . The method of  claim 2 , wherein the mortal disease is selected from the group consisting of cancer, vascular disease and diabetes. 
     
     
         4 . A method of identifying total mutations, wherein the mutations are point mutations for total nonsynonomous point mutations, or total obligatory knockout point mutations or any combination thereof, in any target region of a genome of a population, wherein said point mutations cause or accelerate the appearance of a disease or prevent or delay the appearance of a disease, comprising:
 a) separately determining, in each subpopulation, the set of all detectable mutations occurring at a frequency at or above 5×10 −5  in members of the same population, wherein the subpopulation is selected from the group consisting of young, aged, intermediate age and afflicted with disease; and   b) determining the frequencies of each mutation within the subpopulations, wherein a decrease in the frequency in an afflicted population compared to an unafflicted population of any age is indicative of an allele that causes or accelerates the appearance of a disease, and an increase in the frequency in the afflicted population compared to an unafflicted population of any age is indicative of an allele that prevents or delays the appearance of the disease.   
     
     
         5 . The method of  claim 1 , wherein the control population of individuals not afflicted with the disease is selected from the group consisting of: young, young adult, intermediated aged, aged and mixed aged populations. 
     
     
         6 . The method of  claim 5 , wherein the control population is aged. 
     
     
         7 . The method of  claim 1 , wherein a greater statistically significant sum of the frequencies in the afflicted population relative to the unafflicted population is indicative of a gene that causes or accelerates the disease. 
     
     
         8 . The method of  claim 1 , wherein a decrease in the sum of the frequencies in the afflicted population is indicative of a gene that is protective against the disease. 
     
     
         9 . A method for identifying genes that carry one or more alleles that affect the risk for or age of appearance of a disease, comprising:
 a) identifying and enumerating the set of point mutations that are found in one or more genes or portions thereof of a population not afflicted with the disease, wherein the set comprises all point mutations occurring at a frequency at about or above 5×10 −5  in a population from which the sample was obtained, and determining the frequency with which each point mutation occurs;   b) identifying the set of point mutations that are found in the genes or portions thereof of a population afflicted with the disease, and determining the frequency with which each point mutation occurs; and   c) comparing the frequency of each point mutation identified in a selected gene or portion thereof of the unafflicted population determined in a) with the frequency of the same point mutations identified in the selected gene of the afflicted population determined in b),   wherein a significant difference in the frequency of two or more point mutations in the selected gene of the afflicted population relative to the selected gene of the unafflicted population indicates that the gene one or more alleles that affect the risk for or age of appearance of a disease.   
     
     
         10 . A method for identifying genes that carry one or more alleles that affect the risk for or age of appearance of a disease, comprising:
 a) enumerating the set of point mutations that are found in one or more genes or portions thereof of a population not afflicted with the disease, wherein the set comprises all point mutations occurring at a frequency at about or above 5×10 −5  in a population from which the sample was obtained, and determining the frequency with which each point mutation occurs;   b) identifying the set of point mutations that are found in the genes or portions thereof of a population afflicted with the disease, and determining the frequency with which each point mutation occurs; and   c) comparing the frequency of each point mutation identified in a selected gene or portion thereof of the unafflicted population determined in a) with the frequency of the same point mutations identified in the selected gene of the afflicted population determined in b),   wherein a significant difference in the frequency of two or more point mutations in the selected gene of the afflicted population relative to the selected gene of the unafflicted population indicates that the gene one or more alleles that affect the risk for or age of appearance of a disease.   
     
     
         11 . The method of  claim 1 , wherein the point mutations are detected using a method that comprises:
 a) providing a first pool of DNA fragments comprising a gene or portion thereof, wherein the first pool is isolated from a population and contains DNA pooled from 10 to 10,000 individuals;   b) amplifying a target region of the gene or portion thereof in a high fidelity polymerase chain reaction (PCR) under conditions suitable to produce double-stranded DNA products, wherein the amplified target region comprises a terminal high temperature isomelting domain that is labeled with a detectable label, and wherein a mutant fraction of each PCR-induced mutation among the PCR products is not greater than about 5×10 −5  for a pool created by DNA from 10,000 persons;   c) melting and reannealing the product of b) under conditions suitable to form duplexed DNA, thereby producing a mixture of wild-type homoduplexes and heteroduplexes that contain point mutations;   d) separating the heteroduplexes from the homoduplexes based upon the differential melting temperatures of the heteroduplexes and the homoduplexes and recovering the heteroduplexes, thereby producing a second pool of DNA that is enriched in target regions containing point mutations;   e) amplifying the second pool by high fidelity PCR under conditions where only homoduplexed double-stranded DNA is produced, thereby producing a mixture of homoduplexed DNA comprising wild-type target region and homoduplexed DNA comprising target regions that include point mutations; and   f) separating heteroduplexed DNA comprising target regions that include point mutations based upon the differential melting temperatures of the DNA, and recovering the separated DNA that comprises a target region that includes point mutations,   thereby detecting the point mutations.   
     
     
         12 . The method of  claim 11 , further comprising enumerating the detected point mutations. 
     
     
         13 . The method of  claim 11 , further comprising determining the sequence of the target region of the recovered DNAs to identify point mutations within the target region. 
     
     
         14 . The method of  claim 11 , wherein the first pool of step a) contains DNA pooled from about 100 individuals. 
     
     
         15 . A method for identifying genes that carry one or more alleles that affect the risk for or age of appearance of a disease:
 a) identifying in one or more samples one or more point mutations that are found in one or more genes or portions thereof of a population not afflicted with the disease, determining the frequency with which each point mutation occurs, and calculating the sum of the frequencies of all point mutations identified for a gene or segment thereof;   b) identifying in one or more samples one or more point mutations that are found in one or more genes or portions thereof of a population of a proband population afflicted with the disease, determining the frequency with which each point mutation occurs, and calculating the sum of the frequencies of all point mutations identified for each gene or segment;   c) determining the point mutations that are obligatory knock-out point mutations; and   d) comparing the sum of the frequencies of obligatory knock-out mutations that are found in a selected gene or portion thereof of the unafflicted population calculated in a) with the sum of the frequencies of point mutations that are found in the same gene or portion thereof of the proband population calculated in b),   wherein a significant difference in the sum of the frequencies of point mutations in the proband population indicates that the selected gene carries one or more alleles that affect the risk for or age of appearance of a disease.   
     
     
         16 . A method for identifying genes that carry one or more alleles that affect the risk for or age of appearance of a disease:
 a) determining the number and nucleotide sequence identities of all detectable point mutations in a gene or gene segment in a population not afflicted with the disease, wherein the gene segment comprises exons and splice sites of genes that code for proteins, wherein detectable point mutations are nonsynonomous such as amino acid substitutions, small insertions or deletions, termination codons or splice site mutations, and calculating the sum of frequencies of all point mutations determined for each gene or segment;   b) determining the number and nucleotide sequence identities of all detectable point mutations in a gene or gene segment in a proband population afflicted with the disease, wherein the gene segment comprises of exons and splice sites of genes that code for proteins, wherein detectable point mutations are nonsynonomous such as amino acid substitutions, small insertions or deletions, termination codons or splice site mutations, and calculating the sum of the frequencies of all point mutations determined for each gene or segment;   c) comparing the sum of the frequencies of the specified type or types of nonsynonomous point mutations that are found in a selected gene or segment thereof of the unafflicted population calculated in a) with the sum of the frequencies of the specified type or types of nonsynonomous point mutations that are found in the same gene or segment thereof of the proband population calculated in b),   wherein a statistically significant difference in the sum of the frequencies of the specified type or types of nonsynonomous point mutations associated with a particular gene in the proband population indicates that the selected gene carries one or more alleles that affect the risk for or age of appearance of a disease.   
     
     
         17 . The method of  claim 4 , wherein the point mutations are identified by amplifying DNA obtained from the individual members of the population samples and determining the nucleotide sequence of the DNA.