EP1155361A2 - Procedes visant a reduire la variance des etudes de traitements au moyen de genotypage - Google Patents

Procedes visant a reduire la variance des etudes de traitements au moyen de genotypage

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Publication number
EP1155361A2
EP1155361A2 EP99965095A EP99965095A EP1155361A2 EP 1155361 A2 EP1155361 A2 EP 1155361A2 EP 99965095 A EP99965095 A EP 99965095A EP 99965095 A EP99965095 A EP 99965095A EP 1155361 A2 EP1155361 A2 EP 1155361A2
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European Patent Office
Prior art keywords
control
treated
polymoφhic
treatment
population
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EP99965095A
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German (de)
English (en)
Inventor
Hugh Y. Rienhoff, Jr.
Hywel B. Jones
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DNA Sciences Inc
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Kiva Genetics Inc
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Publication of EP1155361A2 publication Critical patent/EP1155361A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention resides in the fields of medicine, genetics and statistics.
  • Polymorphisms refer to the coexistence of multiple forms of a sequence in a population.
  • a restriction fragment length polymorphism means a variation in DNA sequence that alters the length of a restriction fragment (see, e.g., Botstein et al., Am. J. Hum. Genet. 32:314-331 (1980)).
  • Short tandem repeats are short tandem repeats that consist of tandem di-, tri- and tetra-nucleotide repeat motifs.
  • Such polymorphisms are also sometimes referred to as variable number tandem repeat (VNTR) polymorphisms (see, e.g., U.S. Patent No. 5,075,217; Armour et al., FEBS Lett. 307:113- 115 (1992); and Horn et al., WO 91/14003).
  • VNTR variable number tandem repeat
  • polymorphisms are those involving single nucleotide variations between individuals of the same species; such polymorphisms are called single nucleotide polymorphisms, or simply SNPs.
  • SNPs that occur in protein coding regions give rise to the expression of variant or defective proteins, and thus are potentially the cause of a genetic disease. Even SNPs that occur in non-coding regions can nonetheless result in defective protein expression (e.g., by causing defective splicing). Other SNPs have no phenotypic effects.
  • Certain methods of the invention are designed to provide an assessment of the efficacy of a treatment procedure.
  • such methods involve selecting treated and control subpopulations from treated and control populations of subjects, wherein the treated population has been treated with a treatment procedure and the control population has been treated with a control procedure.
  • the subjects in both the treated and control populations have been characterized for polymorphic profile and are selected because they have similar polymorphic profiles.
  • a determination is then made whether there is a statistically significant difference in a test parameter between the treated and control subpopulations.
  • such a statistically significant difference indicates that there is a correlation between the type of treatment and one or more polymorphic forms within the polymorphic profile for which the treated and control subpopulations were selected.
  • the selecting and determining steps are repeated one or more times.
  • the polymorphic profile for which the treated and control groups are selected differs from the polymorphic profile selected for in previous cycles.
  • the polymorphic profile for which the subpopulations are selected can vary in terms of numbers of polymorphic forms within the profile and the extent of similarity in profiles between treated and control groups.
  • the polymorphic profile can include a single polymorphic form, but more typically includes a plurality of polymorphic forms (e.g., 10 or up to 100 polymorphic forms or more).
  • the polymorphic profiles of the subpopulations have at least 10%, 50%, 75% or up to 100% identity.
  • Certain methods of the invention are directed towards methods for performing clinical trials. Some of these methods initially involve treating a treated population and a control population of patients having the same disease with a drug and a control procedure (e.g., treating with a placebo or with a different amount of the drug or according to a different treatment schedule), respectively. A subpopulation of patients is then selected from each of the treated and control populations for similarity in a polymorphic profile. A determination is then made whether treatment with the drug correlates with status of the disease in the subpopulations to assess the efficacy of the drug in treating the disease. With these methods too, a correlation indicates that at least one or more polymorphic forms within the polymorphic profiles correlates with treatment efficacy. Some methods of the invention are computerized methods.
  • certain methods of the invention include providing a database capable of storing: (1) designations for each member of a treated population treated according to a treatment procedure and designations for each member of a control population treated according to a control procedure, (2) designations for a polymorphic profile for each member of the treated and control populations, and (3) designations for a test parameter for each member of the treated and control populations.
  • a database capable of storing: (1) designations for each member of a treated population treated according to a treatment procedure and designations for each member of a control population treated according to a control procedure, (2) designations for a polymorphic profile for each member of the treated and control populations, and (3) designations for a test parameter for each member of the treated and control populations.
  • subpopulations from each of the treated and control populations are selected for similarity in polymorphic profile.
  • a determination is then made to ascertain whether there is a statistically significant difference in the test parameter between the subpopulations.
  • the output from the determining step is then displayed on an output device (e.g.,
  • the invention provides various computer systems and programs. For instance, certain computer products for assessing a treatment procedure are provided. Some systems include program products that generally include code for providing or receiving data, wherein the data includes: (1) designations for each member of a treated population treated according to a treatment procedure and for each member of a control population treated according to a control procedure, (2) designations for a polymorphic profile for each member of the treated and control populations, and (3) designations for a test parameter for each member of the treated and control populations.
  • the program also includes code for selecting a subpopulation from each of the treatment and control populations that have a similar polymorphic profile, code for determining whether there is a statistically significant difference in the test parameter between the subpopulations and code for displaying an output that indicates whether a statistically significant difference was found between the subpopulations.
  • the code is typically stored on a computer readable storage medium.
  • the invention further provides a computerized system for assessing treatment procedures.
  • Some systems generally include a memory, a system bus and a processor.
  • the processor is operatively disposed to provide or receive data, wherein the data includes: (1) designations for each member of a treated population having been treated according to a treatment procedure and for each member of a control population treated according to a control procedure, (2) designations for a polymorphic profile for each member of the treated and control populations, and (3) designations for a test parameter for each member of the treated and control populations.
  • the processor is further disposed to select a subpopulation from each of the treatment and control populations that have a similar polymorphic profile and determine whether there is a statistically significant difference in the test parameter between the subpopulations.
  • the microprocessor is also capable of displaying an output indicating whether a statistically significant difference was found between the subpopulations.
  • FIG. 3 is a flow chart for a method of assessing a treatment procedure according to the present invention.
  • a “treatment procedure” refers to methods or processes that are performed on a member of a treated or treatment population.
  • the treatment procedure is a process performed on a subject to affect some biological condition, susceptibility, or resistance of the subject.
  • treatment procedures include, but are not limited to, treatment with pharmaceutical compounds or other biologies (including, for example, recombinantly produced proteins), surgical procedures and various behavioral therapies (e.g. , prescribed diet and/or exercise regimes).
  • Treatment procedures can be prophylactic or therapeutic.
  • a treatment procedure can include treating members of a treatment population with a vaccine.
  • a "control procedure” refers to methods that are performed on a member of a control population.
  • the control procedure can differ from the treatment procedure in quantitative or qualitative aspects.
  • the members of a treatment population can receive a pharmaceutical composition, whereas the control population receives a placebo (i.e., no pharmaceutical composition).
  • the control procedure involves administration of a drug at different concentrations than the treatment procedure or can involve a different schedule for administering the pharmaceutical composition relative to the treatment procedure.
  • a "clinical study” is an inquiry into the cause and sometimes treatment of a particular phenotype that is represented by at least one random variable. This phenotype may often be a disease state or a measure of disease severity.
  • a clinical study can take the form of a case-control study (for a discrete random variable, the groups being affected and unaffected individuals) or a single population study where the cause of the degree or severity of the phenotype is being investigated (for example, a quantitative study can examine blood pressure, blood glucose, etc.).
  • a “treatment study” is an inquiry into the effect or influence a particular treatment procedure has on a biological condition, biological susceptibility or biological resistance of a subject. The study can be quite structured, formal and extensive in scope, or can be relatively unstructured and of limited scope.
  • a treatment study can be a formal clinical trial or study performed on a relatively large group of subjects wherein the study is performed according to set guidelines (e.g., governmental regulations).
  • the treatment study can also be a preclinical study, a field trial of a plant population or even an informal study by a scientist, veterinarian or a physician of the effects of a treatment on relatively few subjects.
  • the subjects are divided into several (though often just two) groups. These may represent different doses ranges or simply the treated and the untreated subjects.
  • the random variable is measured after treatment. It may also be measured before treatment if it is a change in the variable over time that is being investigated (e.g., bone mineral density or blood pressure).
  • subjects are not undergoing any other treatments for their pathological condition. However, if such a constraint is unreasonable, the study should be designed so that subjects in both treated and untreated groups are undergoing the same alternative treatment.
  • the subjects of the treatment study can be conducted with any type of organism, including, for example, animals (including humans), plants, bacteria and viruses.
  • a “biological condition” refers to the condition, susceptibility or resistance of the organism upon which the study seeks to determine whether the treatment procedure has an effect.
  • the biological condition is a physical or physiological condition of the organism.
  • the biological condition is a pathological condition (i.e., a physiological state that normally does not exist, such as a disease for example).
  • Pathological conditions typically studied with the methods of the invention are those with a minimal environmental variance (e.g., high cholesterol levels in serum), although this is not required. Examples of pathological conditions include AIDS, arteriosclerosis, cancer, and diabetes, elevated blood pressure, elevated serum cholesterol level or psychosis.
  • a biological condition can be the biological susceptibility or resistance of a subject.
  • the treatment study can involve an analysis of the effect of certain treatments on the susceptibility of a plant to an herbicide or susceptibility of a plant to frost damage.
  • the study can be directed towards an organism defense response (i.e., resistance) to some type of insult, for example.
  • a "random variable,” either discrete or continuous, can be any biological, physiological or biochemical endpoint measured or observed, particularly in the setting of a clinical study. This includes measured and observed effects of treatments, the changes in those observations and measurements over the course of time (the so -called natural history), or any other intervention that may alter traits, signs or symptoms.
  • random variables include pathological conditions susceptible to treatment with, e.g., pharmaceutical compounds; biologies, including recombinantly produced proteins; surgical techniques; restrictive diets; and behavioral therapy. For example, serum concentrations of cholesterol, height, body mass, are all continuous random variables.
  • This notion can extend to discrete variables such as the presence or absence of a physical trait or symptom which include, for example, nevi on the skin, cysts in the liver, particular antibodies in the serum, the degree of swollenness of the joints, the number of affected joints, or the number of hallucinations in a psychotic episode.
  • test parameter is the characteristic that is measured or observed to determine the effect or efficacy (or lack thereof) of the treatment procedure being evaluated and is utilized to determine whether there is a statistically significant difference in the treatment and control protocols.
  • the test parameter can be a random variable. Typically, the test parameter is expressed in quantitative terms, although in some instances the test parameter can be evaluated in qualitative terms.
  • the nature of the test parameter varies according to the biological condition being studied. If the biological condition is a disease, the test parameter provides a measure for the status of the disease. For example, if the biological condition is AIDS, the test parameter can be the concentration of HIV in the blood of a subject. If the biological condition is arteriosclerosis, the test parameter can be serum cholesterol concentration.
  • variance refers to variation, scatter, spread or dispersion about the arithmetic mean.
  • the variance is the mean value of the squared deviations (Armitage, P., STATISTICAL METHODS IN MEDICAL RESEARCH, Blackwell Scientific, Oxford, United Kingdom (1971)).
  • a large variance indicates large deviations from the arithmetic mean. For example, if cholesterol level is the test parameter being measured, a mean cholesterol level is determined. The variance represents the average squared deviation of all cholesterol levels relative to the mean. Other statistical measures of spread or dispersion about a mean can also be used.
  • the distribution of the test parameter takes the shape of a bell-shaped or a normal (Gaussian) curve. Pictorially, the invention decreases the variance and thus narrows the bell-shape of the normal curve or, described mathematically, the distribution becomes leptokurtic.
  • the variance is due to dissimilar effects on the subjects that influence the biological condition being analyzed by statistical methods, e.g., genetic, environmental and measurement variables.
  • statistical methods e.g., genetic, environmental and measurement variables.
  • genetic differences between individual subjects and the environment in which the subjects live. Examples of environmental influences include diet, sleep patterns, geographical location and culture.
  • a polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population generally said to be occurring at a frequency of greater than 0.1 %.
  • a polymorphic marker or site is the locus at which genetic divergence occurs.
  • Preferred markers have at least two alleles, each occurring at frequency of greater than 1% in a selected population.
  • a polymorphic locus can be as small as one base pair. Such a locus is referred to as a single nucleotide polymorphism or simply SNP.
  • Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • the first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles.
  • the allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form or allele and the other forms referred to as mutant forms or alleles. Diploid organisms can be homozygous or heterozygous for allelic forms.
  • a diallelic polymo ⁇ hism has two forms.
  • a triallelic polymo ⁇ hism has three forms.
  • a "single nucleotide polymo ⁇ hism" occurs at a polymo ⁇ hic site that is occupied by a single nucleotide, which is the site of variation between allelic sequences.
  • the site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
  • a single nucleotide polymo ⁇ hism usually arises due to substitution of one nucleotide for another at the polymo ⁇ hic site.
  • a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine or vice versa.
  • Single nucleotide polymo ⁇ hisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • a "polymo ⁇ hic profile” refers to one or more polymo ⁇ hic forms for which a subject is characterized.
  • a polymo ⁇ hic form is characterized by identifying which nucleotide(s) is (are) present at a polymo ⁇ hic site in a nucleic acid sample acquired from a subject.
  • the profile includes at least one polymo ⁇ hic form and preferably includes a plurality of polymo ⁇ hic forms, such as at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 polymo ⁇ hic forms or more.
  • Polymo ⁇ hic profiles are similar when the polymo ⁇ hic profiles being compared share at least one polymo ⁇ hic form at least one polymo ⁇ hic site.
  • polymo ⁇ hic profiles share identity of polymo ⁇ hic forms in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in at least 10, 20, 30, 40, 50, 60, 70, 100, or 500 polymo ⁇ hic sites.
  • Polymo ⁇ hic forms are identical if the nucleotide(s) at a particular polymo ⁇ hic site are the same.
  • two polymo ⁇ hic profiles each including 10 polymo ⁇ hic forms are 50% identical if five of the polymo ⁇ hic forms in the two profiles are identical.
  • the polymo ⁇ hic forms at each polymo ⁇ hic site are considered to be identical in two individuals if both individuals have the same two alleles at the polymo ⁇ hic site.
  • an individual having alleles al and a2 at polymo ⁇ hic site A is considered to have the same profile as an individual having alleles al and a2 but not to an individual having alleles al and al, or a2 and a2, or al and a3 and so forth.
  • linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome, and can be measured by percent recombination between the two genes, alleles, loci or genetic markers.
  • Linkage disequilibrium or "allelic association” means the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance (see, for example, Weir, B., Genetic Data Analysis, Sinauer Associate Inc., 1996). For example, if locus X has alleles a and b, which occur equally frequently, and linked locus Y has alleles c and d, which occur equally frequently, one would expect the combination ac to occur with a frequency of 0.25. If ac occurs more frequently, then alleles a and c are in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium with linked alleles.
  • a marker in linkage disequilibrium can be particularly useful in detecting susceptibility to disease (or other phenotype) notwithstanding that the marker does not cause the disease.
  • a marker (X) that is not itself a causative element of a disease, but which is in linkage disequilibrium with a gene (including regulatory sequences) (Y) that is a causative element of a phenotype can be detected to indicate susceptibility to the disease in circumstances in which the gene Y may not have been identified or may not be readily detectable.
  • “Haplotype” refers to a collection of polymo ⁇ hic markers either in close physical proximity on a single chromosome, or unlinked physically but associated together, which confers a biologic property or association with a phenotype.
  • nucleic acid is a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, including known analogs of natural nucleotides unless otherwise indicated.
  • the term "primer” refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair means a set of primers including a 5' upstream primer that hybridizes with the 5 1 end of the DNA sequence to be amplified and a 3', downstream primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • nucleic acid probe refers to a nucleic acid molecule that binds to a specific sequence or sub-sequence of another nucleic acid molecule.
  • a probe is preferably a nucleic acid molecule that binds through complementary base pairing to the full sequence or to a sub-sequence of a target nucleic acid.
  • Probes can bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are typically directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex can later bind.
  • a "label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., by inco ⁇ orating a radio-label into the peptide, and used to detect antibodies specifically reactive with the peptide).
  • a label often generates a measurable signal, such as radioactivity, fluorescent light or enzyme activity, which can be used to quantitate the amount of bound label.
  • a "labeled nucleic acid probe” is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe can be detected by detecting the presence of the label bound to the probe.
  • sequenceselectively hybridizes to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent hybridization conditions refers to conditions under which a probe hybridizes to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • Tm thermal melting point
  • Stringent conditions are those in which the salt concentration is less than about 1.0 sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions can also be achieved with the addition of destabilizing agents as formamide. If degenerate hybridization is desired, less than stringent conditions are necessary. For example, if single nucleotide mismatching is preferred, hybridization conditions will be relaxed with lower temperatures and higher salt content.
  • statistal correlation refers to a statistical association between two variables or parameters as measured by any statistical test including, for example, chi-squared analysis, ANOVA or multivariate analysis.
  • the correlation between a polymo ⁇ hic form of DNA and a random variable or test parameter is considered statistically significant if the probability of the result happening by chance (the P-value) is less than some predetermined level (e.g., 0.05).
  • the term “statistically significant difference” refers to a statistical confidence level, P, that is ⁇ 0.05, preferably ⁇ 0.01, and most preferably ⁇ 0.001.
  • a “drug” or “pharmaceutical agent” means any substance used in the prevention, diagnosis, alleviation, treatment or cure of a disease.
  • the terms include a vaccine, for example.
  • Tissue means any sample taken from any subject, preferably a human. Tissues include blood, saliva, urine, biopsy samples, skin or buccal scrapings, and hair.
  • patient refers to both human and veterinary subjects.
  • the present invention provides methods, computer programs and computerized systems useful for designing treatment studies and for evaluating the efficacy of various types of treatment procedures (e.g., clinical trials) as a function of the genotype of a subject.
  • the methods of the invention are designed to control for underlying genetic factors that may influence the response to a treatment.
  • the present invention is based, in part, on the insight that controlling, either directly or indirectly, genetic factors that influence a patient's response to treatment can greatly increase the power of the clinical trial.
  • Some methods are designed to reduce the genetic diversity of the patient population so as to increase the probability of individuals sharing the same alleles at genes involved in response to the treatment. In cases where polymo ⁇ hisms (usually in genes) are known to be associated with or cause differences in response to the treatment, these polymo ⁇ hisms can be used directly in the design of the clinical trial.
  • the invention provides methods for reducing the variance in the biological condition or phenotype of interest by controlling for genetic factors influencing that phenotype.
  • the phenotype of interest is the response to a treatment.
  • Genetic factors can be controlled in a number of different ways but the principle underlying the methods of the invention can be illustrated by an example.
  • test parameter is measured in two groups, the first (which is of size ⁇ ) is treated and the second (of size m) is untreated, the mean and variance of these samples can be calculated in the standard way (see Armitage & Berry, Statistical Methods in Medical Research, Blackwell Science, 1995.)
  • the mean and variance of the treated group are ⁇ i and sf respectively
  • the mean and variance of the untreated group are ⁇ 2 and s 2 2 , respectively.
  • Z al2 is the value of the standard normal distribution that is exceeded by chance in a 12% of cases.
  • any method that decreases the variance in either sample i.e., which decreases _? 2 or ) necessarily decreases the size of the confidence interval.
  • the size of the confidence interval can be held constant with fewer patients enrolled in the trial (i.e., n and/or m can be reduced).
  • reducing the variance in response can lead either to greater certainty of a difference (here encapsulated by a smaller confidence interval) or in a reduced sample size for the same statistical power.
  • the variance can be reduced in a number of different ways as described in the following sections.
  • a set of polymo ⁇ hic markers can be examined in a large group of subjects and those with similar polymo ⁇ hic profiles enrolled in the treatment study. Inco ⁇ orating genetic factors (represented by the polymo ⁇ hic profile) into the inclusion/exclusion criteria of a treatment study allows an experimenter to reduce the variance in response due to underlying genetic factors.
  • a second approach is to categorize individuals into subsets depending on how similar the polymo ⁇ hic profiles are to one another. Within each subset, subjects are randomly allocated into treatment or control subpopulations, as they are in a standard clinical trial for example. This method of dividing the subjects creates subsets that are genetically more homogenous than a random sample of the same size. This design is equivalent to conducting several small, independent treatment studies each of which contains patients that have more similar polymo ⁇ hic profiles than expected by chance. Many environmental variables can be manifestations of underlying genetic factors.
  • stratification refers to the division of the sample into subsets that are more similar than expected by chance for a given factor.
  • those known to be non-responders by their polymo ⁇ hic profile can be treated according to a control procedure (e.g., administered a placebo), while those who deemed responders from their polymo ⁇ hic profile can be given the treatment procedure (e.g., administered a drug).
  • a control procedure e.g., administered a placebo
  • those who deemed responders from their polymo ⁇ hic profile can be given the treatment procedure (e.g., administered a drug).
  • the treatment procedure e.g., administered a drug
  • this information may be used to allocate the most appropriate dose to subjects enrolled in a treatment study such as a clinical trial.
  • the polymo ⁇ hic profiles of patients can determine the degree of response of individuals to the treatment. In this way, it may be possible to allocate different doses to different patient depending on their polymo ⁇ hic profiles. For example, if a treatment potentially has side effects, it will be desirable to administer the minimum efficacious dose. This can vary for subjects with different polymo ⁇ hic profiles.
  • Data obtained from such a treatment study are re-analyzed on subsets of the treated and control populations selected for similarity of a polymo ⁇ hic profile to each other.
  • the reanalysis of data is carried out on subsets of individuals sharing a similar polymo ⁇ hic profile and indicates whether the treatment reaches statistical significance on individuals having that profile. If the profile contains one or more polymo ⁇ hic forms associated in some way with the biological condition of interest (e.g., disease), the treatment may reach statistical significance on the subpopulations when it does not on the initial treatment populations. If the profile does not contain such polymo ⁇ hic DNA forms, then the re-analysis of data also shows a lack of statistical significance.
  • a further re-analysis is performed in which further subpopulations of individuals from treated and control populations are selected for similarity to a second polymo ⁇ hic profile. Because the individuals have already been characterized for polymo ⁇ hic profile, the second re-analysis can be performed without further experimental work in a highly automated and iterative fashion. Again, the second analysis indicates whether the treatment reaches statistical significance on the individuals having similarity to the polymo ⁇ hic profile by which subpopulations are selected in the second analysis.
  • a suitably programmed computer can perform thousand, millions or billions of cycles of analysis in which different subpopulations of individuals are selected based on similarity to different polymo ⁇ hic profiles. Performing multiple tests typically requires a re-evaluation of the p-value at which a result is declared to be statistically significant to control the rate of false positive results. If after exhaustive analysis, statistical significance is not reached for any polymo ⁇ hic profile, one can conclude with increased confidence that the treatment procedure (e.g., administration of a drug) being tested is unlikely to be effective in any significant portion of the population, and that further research is not justified.
  • the treatment procedure e.g., administration of a drug
  • a clinical trial can be carried out as follows:
  • polymo ⁇ hisms A set of polymo ⁇ hisms is identified that allow the division of the patient cohort into sub-groups. These polymo ⁇ hisms may be known to be involved in the test parameter (e.g., the phenotype or endpoint) that is to be measured or can be chosen at random. (In the latter case, the genetic sub-groups may show identical results with respect to the phenotype of interest. This implies the method of grouping does not decrease the variance in the endpoint and the population can be re-analyzed as a whole. Thus, stratification by using genetic data does not have a deleterious effect on the experiment or trial, even in cases where it does not influence the outcome).
  • test parameter e.g., the phenotype or endpoint
  • Genotyping of the cohort Some or all of the markers are genotyped in the entire cohort of patients enrolled in the clinical trial. These data are then used either as inclusion/exclusion criteria (see 3a below) or to divide the cohort into subgroups (see 3b below).
  • 3b Division of the clinical trial into subgroups.
  • a metric is used to determine the genetic similarity of patients in the cohort. This information is used to divide the population into subgroups that have greater genetic similarity than might be expected by chance. That is, the subgroups are Q genetically more homogenous than a random subset of the same size.
  • the precise method of measuring similarity will depend on the number and type of markers used. In the simplest case, the number of markers at which two individuals have the same alleles can be used to determine similarity. Many other more complex metrics can be employed that, for example, giving extra weight to markers 5 known to be particularly informative or that influence the test parameter of interest.
  • an experimenter can control the number of subgroups that need to be formed. For N individuals, this can range from 1 (the entire population) to N (each individual is in a separate subgroup). Practical as well as scientific reasons are considered in determining how many subgroups 0 are optimal for a given experiment or trial. With the methods of the invention, groups can be merged at a later time.
  • One method is to randomize the treatment and placebo within each subgroup. This is similar to treating each subgroup as a separate experiment or clinical trial. Results of each subgroup may be analyzed separately or may be pooled and then 0 analyzed. Alternatively, treatment can be non-randomly allocated within the subgroups. This may be appropriate, for example, when the polymo ⁇ hisms are known to be associated with the outcome or endpoint of interest. For example, in the context of a clinical trial, if there are only two subgroups and one of the subgroups is known to contain high responders and the other low responders to a treatment, allocating the treatment to the first group and the placebo to the second group maximizes the difference between response for treated and untreated individuals. Conversely, allocating the placebo to the first group and the treatment to the second group shows the minimum difference between treated and untreated individuals. Which of these approaches is most appropriate depends on the exact objective of the experiment or clinical trial.
  • the utility of stratifying by using a set of genetic polymo ⁇ hisms can be re-assessed through successive experiments of clinical trials. Uninformative polymo ⁇ hisms can be dropped and new polymo ⁇ hisms added to increase the usefulness of the set as a whole. Use of these polymo ⁇ hisms in subsequent treatment studies or clinical trials leads to greater reproducibility of results and the need for enrolling fewer subjects in replication studies.
  • a clinician can devise clinical trials that involve fewer subjects, decrease the confidence intervals, or increase the precision or discriminatory power of a given trial. The clinician can decide which of these three aspects of trial design or analysis to change while keeping the other two constant.
  • polymo ⁇ hic markers in a clinical trial population permits, upon analysis, the identification of subsets of polymo ⁇ hic markers that may correlate with either a salubrious response, unresponsiveness or excessive response to a treatment, an unwanted or toxic response to a treatment, and may identify by virtue of unresponsiveness, a clinical subset of patients that define a "different" disease.
  • apost facto genetic analysis correlated with a specific clinical phenotype such as drug responsiveness or unresponsiveness can reveal different etiologic mechanisms for the disease being treated.
  • phenotypic markers can provide insight into genetic diversity of the subjects being treated allowing the clinician to alter enrollment in a drug trial to accommodate more or less genetic diversity as is scientifically prudent.
  • members of a treated and control (untreated) population having a biological condition of interest are characterized for polymo ⁇ hic profile and a test parameter that is a measure of the biological condition, assuming the members have not already been so characterized.
  • the members in the treated population have been (or are) treated according to a treatment procedure, whereas the members of the control population have been (or are) treated according to a control procedure.
  • subpopulations from the treated and control populations are selected for similar genetic composition such that the members in the two populations have similar or identical polymo ⁇ hic profiles.
  • the polymo ⁇ hic profile of the subpopulations includes one or more polymo ⁇ hic forms.
  • the polymo ⁇ hic profile includes a plurality of polymo ⁇ hic forms, generally at least 5, in other instances at least 10, and in still other instances at least 100, or any number there between.
  • the polymo ⁇ hic profiles for the two groups are selected to be similar. This means that there is at least one common polymo ⁇ hic form between the two subpopulations, although typically there are more.
  • the polymo ⁇ hic profiles for the two subpopulations are typically at least 10% identical, in some instances at least 50% identical, in still other instances greater than 75% identical, in yet other instances 90% identical or more, and in still other instances 100% identical.
  • the analysis of phenotypic markers can provide additional insight into the genetic diversity of the subjects being treated and allows the researcher to alter the members in a study to accommodate more or less genetic diversity.
  • Polymo ⁇ hisms can be selected in three distinct ways. First, they can be chosen at random. Second, only those polymo ⁇ hisms known or suspected to be involved in the phenotype of interest (response to treatment in the case of a clinical trial) can be selected. Third, DNA polymo ⁇ hism selection can be driven by identifying polymo ⁇ hisms that reside in regions of the genome that have previously been shown to harbor a genetic linkage with the observable trait(s) under study. In the first case, random polymo ⁇ hisms are unlikely to be directly involved in response. However, they can be used to determine the genetic similarity of patients and hence can be used to form subgroups that are more genetically homogenous than expected by chance.
  • This strategy is particularly effective when a large number of (usually unknown) genes are involved in determining an individual's response to treatment. If there are polymo ⁇ hisms known to be involved in response or to be associated with (possibly unknown) genes involved in response, then these can preferentially be used to determine subgroups of patients.
  • polymo ⁇ hisms will be equally informative or useful in determining subgroups. Factors such as the allele frequencies, whether the polymo ⁇ hism is protein coding or non-coding, whether the polymo ⁇ hism is in linkage disequilibrium with another polymo ⁇ hism already in the polymo ⁇ hic profile being used, whether the polymo ⁇ hism is in linkage disequilibrium with a gene or genes known to be involved in response to treatment can all influence the utility of a given polymo ⁇ hism. Note that in some cases, it may be desirable to give more weight to some polymo ⁇ hisms than others in the formation of subgroups. That is, polymo ⁇ hisms known to be associated with responsiveness may be more important (and hence given more weight) than random polymo ⁇ hisms.
  • the polymo ⁇ hisms can be in genomic DNA, RNA or cDNA. While any polymo ⁇ hisms can be used, those of particular import are polymo ⁇ hisms in genes that encode proteins that directly or indirectly influence a biochemical pathway that is correlated with the biological condition being measured or observed. Thus, for example, if a study involves assessing the efficacy of methods for treating patients having elevated blood cholesterol levels, the polymo ⁇ hic profile can be tailored to include polymo ⁇ hisms located in genes known to be involved in cholesterol synthesis and metabolism.
  • a finding of a statistically significant difference indicates that the polymo ⁇ hic forms in the polymo ⁇ hic profile of the treated subpopulation correlate with the biological condition (e.g. , the polymo ⁇ hic profile is correlated with a particular disease) and that the treatment method under study is useful (or not beneficial) for treating subjects with the biological condition.
  • the correlation identifies a set of genetic markers associated with the disease and thus has diagnostic value. In other instances, the correlation identifies markers that are associated with a positive treatment result and thus are important from a therapeutic standpoint.
  • a statistically significant difference in a test parameter between the treatment and control subpopulations can be determined using standard methods of statistical analysis. Methods include, for example, the analysis of variance, logistic regression, cluster analysis, non-parametric statistics, contingency table test and other standard statistical tests.
  • the polymo ⁇ hic profile of the subpopulation initially selected often do not correlate with a statistically significant difference in the test parameter that is used to measure the efficacy of treatment.
  • the method can be repeated with different subpopulations created by using an alternative definition or measure of genetic similarity, or by dividing the population into greater or fewer sub-populations. This reflects the fact that there will rarely be a single unique way to group patients. Indeed, for a study with N individuals, it will often be possible to form any number of sub- populations from 1 (the entire population) to N (each individual in its own sub- population). Repeating the process is often an effective way of detecting which polymo ⁇ hisms within the polymo ⁇ hic profile are particularly informative with respect to the test parameter of interest.
  • additional cycles can be repeated using, for example, a subset of the polymo ⁇ hic forms utilized in an earlier cycle to determine whether the subset might show an even greater correspondence with the test parameter and thus treatment efficacy.
  • the polymo ⁇ hic forms within a polymo ⁇ hic profile evolve over time to account for a greater proportion of the genetic component of the variance.
  • these polymo ⁇ hic forms generally do not contribute equally. Some account for more variance than others; markers that do not correlate with differences in the treatment and control procedures are discarded from the analysis.
  • the set of markers as a collection have value distinct from the individual markers. This collection has enduring value for understanding the genetic contribution to a distinct biological condition of interest. Individual markers can have diagnostic utility, as can the collection.
  • the analysis of treatment or trial data involving groups and subgroups is amenable to both parametric and non-parametric (distribution-free methods) statistical methods.
  • the members of the treatment and control groups all share some biological condition upon which the study is designed to determine whether the treatment procedure has a statistically significant different effect relative to the control procedure.
  • the members of the treatment and control groups can be essentially any type of organism including, for example, humans, non-human animals, plants, bacteria and viruses. In some instances, the members are mammals (e.g., humans, primates) that are part of a clinical trial involving the testing of a pharmaceutical agent or behavioral therapy for example.
  • the number of members in the subpopulations selected from the treatment and control group is at least one but generally is more than one, typically at least 5, in other instances at least 10, and in still other instances at least 100 or more, although any number of members between these numbers can also be selected.
  • the members of the subpopulation are selected not only to have similar polymo ⁇ hic profiles, but also to have other common features. Selecting on the basis of other commonalties can further reduce total variance beyond that achieved by reducing the variance attributable to genetic factors.
  • members of the treatment and control subpopulations can also be selected to have been similarly exposed to an environmental factor. Examples of such environmental factors include, but are not limited to, exposure to various agents such as radiation, chemicals, and second hand smoke; geographical location; and life style characteristics such as sleep patterns, diet, and amount of exercise.
  • the types of treatment and control procedures vary according to the biological condition to which the treatment is directed.
  • the biological conditions can be any of a number of conditions, such as a pathological condition or simply a biological susceptibility, for example.
  • a variety of different procedures can be performed when the biological condition is a pathological condition.
  • the procedures involve administering a pharmaceutical agent, including, for example: 1) administering a pharmaceutical agent to members of the treated population and giving members in the control population a placebo or nothing at all, 2) giving members of the treated population one pharmaceutical agent (or combination of pharmaceutical agents) and a different pharmaceutical agent (or combination of pharmaceutical agents) to the control members; 3) providing one quantity of a pharmaceutical agent to the treated population and a different amount to the control population, or 4) administering a pharmaceutical agent to the treatment and control populations according to different schedules.
  • a pharmaceutical agent including, for example: 1) administering a pharmaceutical agent to members of the treated population and giving members in the control population a placebo or nothing at all, 2) giving members of the treated population one pharmaceutical agent (or combination of pharmaceutical agents) and a different pharmaceutical agent (or combination of pharmaceutical agents) to the control members; 3) providing one quantity of a pharmaceutical agent to the treated population and a different amount to the control population, or 4) administering a pharmaceutical agent to the treatment and control populations according to different schedules.
  • the treatment procedure can include some type of behavioral therapy.
  • behavioral therapy include, but are not limited to, a particular diet regime (e.g., low fat, low sodium, high protein, or a restricted calorie diet), a prescribed exercise regime (e.g., exercising for a certain time period a certain number of times a week, performing low-impact exercises, exercising to reach a target heart rate, therapies that work certain muscle groups), meditation, yoga, and stress reduction techniques.
  • a particular diet regime e.g., low fat, low sodium, high protein, or a restricted calorie diet
  • a prescribed exercise regime e.g., exercising for a certain time period a certain number of times a week, performing low-impact exercises, exercising to reach a target heart rate, therapies that work certain muscle groups
  • meditation yoga
  • stress reduction techniques e.g., the treatment procedure can include combinations of the foregoing procedures as well.
  • Members in control groups may not undergo therapy at all or may be treated in opposing fashion (or may already be engaged in contrary behaviors). For example,
  • the treatment procedure can also be directed towards a biological susceptibility or resistance rather than a pathological condition.
  • plants can be treated with various agricultural agents used to affect plant growth or health (e.g., fertilizer or other growth stimulants, herbicides, insecticides, and pH altering agents) to assess the effect of such agents on various susceptibilities or resistances of plants (e.g., susceptibility to frost or freeze damage and resistance to herbicides).
  • various agricultural agents used to affect plant growth or health e.g., fertilizer or other growth stimulants, herbicides, insecticides, and pH altering agents
  • humans or other organisms can also be treated with various agents, for example vaccines, to determine the effect of the agents on various susceptibilities or resistances.
  • the treatment methods described herein permit the identification of subsets of polymo ⁇ hic forms that correlate with either a favorable response or unresponsiveness to treatment, or an unwanted or toxic response to a treatment.
  • Clinical trials on the efficacy of certain pharmaceutical treatments can identify individuals that are unresponsive to treatment and, in so doing, can in some instances result in the identification of a clinical subset of patients that define a "different" disease.
  • Such correlations can also be used as a prognostic and/or diagnostic tool to identify subjects having or likely to acquire a disease or to select appropriate treatment procedures for a subject based upon the particular genetic composition of the subject.
  • Information gained from clinical trials in which patients are genotyped for a set of polymo ⁇ hic genetics markers can also be used in other stages of drug discovery and development. For example, genes shown to be associated with response via the polymo ⁇ hic profile of the patients may be amenable to intervention and hence represent potential drug targets. Furthermore, identification of treatments that show low efficiency (i.e., many non-responders) or that have high rates of adverse events can be identified by examining the polymo ⁇ hism profile of patients in early phase trials. This information can then be used in the decision whether to take a treatment forward into large and more costly trials. For example, if non-response is associated with a polymo ⁇ hism profile that is common in the general population, it may be inappropriate to use the treatment in a larger trial.
  • FIG. 1 depicts a representative computer system 10 suitable for implementing certain methods of the present invention.
  • computer system 10 typically includes a bus 12 that interconnects major subsystems such as a central processor 14, a system memory 16, an input/output controller 18, an external device such as a printer 23 via a parallel port 22, a display screen 24 via a display adapter 26, a serial port 28, a keyboard 30, a fixed disk drive 32 via storage interface 34, and a floppy disk drive 33 operative to receive a floppy disk 33 A.
  • Many other devices can be connected such as a scanner 60 via I/O controller 18, a mouse 36 connected to serial port 28, a CD ROM player 40 operative to receive a CD ROM 42, or a network interface 44.
  • Source code to implement the present invention can be operably disposed in system memory 16 or stored on storage media such as a fixed disk 32 or a floppy disk 33 A.
  • Other devices or subsystems can be connected in a similar manner. All of the devices shown in FIG. 1 are not required to practice the present invention.
  • the devices and subsystems can also be interconnected in different ways from that shown in FIG. 1.
  • the operation of a computer system 10 such as that shown in FIG. 1 is readily known in the art; hence, operations of the system are not described in detail herein.
  • FIG. 2 is an illustration of a representative computer system 10 of FIG. 1 suitable for performing the methods of the present invention; however, FIG. 2 depicts but one example of many possible computer types or configurations capable of being used with the present invention.
  • computer system 10 can include display screen 24, cabinet 20, keyboard 30, a scanner 60, and mouse 36.
  • Mouse 36 and keyboard 30 are examples of "user input devices.” Other examples of user input devices include, but are not limited to, a touch screen, light pen, track ball and data glove.
  • Mouse 36 can have one or more buttons such as buttons 37.
  • Cabinet 20 houses familiar computer components such as floppy disk drive 33, a processor 14 and a storage means (see FIG. 1).
  • Cabinet 20 can include additional hardware such as input/output (I O) interface for connecting computer system 10 to external devices such as a scanner 60, external storage, other computers or additional peripheral devices.
  • system 10 includes a computer having a Pentium® microprocessor 14 that runs WINDOWS® Version 3.1, WLNDOWS95® or WINDOWS98® operating system by Microsoft Co ⁇ oration.
  • Pentium® microprocessor 14 that runs WINDOWS® Version 3.1, WLNDOWS95® or WINDOWS98® operating system by Microsoft Co ⁇ oration.
  • the methods of the invention can easily be adapted to other operating systems (e.g. , UNIX) without departing from the scope of the present invention.
  • FIG. 3 is a flowchart of simplified steps in one computerized method of the invention for assessing a treatment procedure.
  • a database is provided that contains a plurality of designations for each member of a population that has either been treated according to a treatment procedure or a control procedure and that shares a common biological condition (the database is described further below).
  • the population includes both treatment and control populations.
  • One group of designations is to identify each member of the two populations.
  • Subpopulations from the treated and control populations are selected in a selection step 102 for similarity in polymo ⁇ hic profile.
  • determining step 104 a determination is made whether there is a statistically significant difference in the test parameter between the subpopulations. A statistically significant difference indicates that the polymo ⁇ hic profile of the subpopulations correlates with the biological condition and effect of treatment.
  • a displaying step 106 an output of the result of the determining step is displayed on an output device to facilitate the analysis.
  • the selecting step 102, the determining step 104, and the displaying step 106 are repeated using subpopulations that have a polymo ⁇ hic profile that is different from that in earlier cycles.
  • the microprocessor in the computer system of the present invention is operatively disposed relative to the system memory, the system bus and the input/output so as to perform the foregoing functions.
  • the processor provides or receives data that comprises designations for each member of the treated and control populations, as well as designations for a polymo ⁇ hic profile and a test parameter for each member of the two populations.
  • the microprocessor is also operatively disposed to select a subpopulation from each of the treatment and control populations for similarity in polymo ⁇ hic profile, determine whether there is a statistically significant difference in the test parameter between the subpopulations and display an output of the result obtained.
  • the computer program of the invention includes code for providing or receiving data comprising the various designations for the identity of the members of the test and control populations, their polymo ⁇ hic profiles and test parameter results.
  • the program also includes code necessary to perform the selecting, determining and displaying steps set forth above.
  • Polymo ⁇ hisms are detected in a target nucleic acid from an individual being analyzed.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
  • the target nucleic acid is a cDNA encoding cytochrome P450
  • the liver is a suitable source.
  • Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • A. Detection of Polymo ⁇ hisms in Target DNA There are two distinct types of analysis depending whether a polymo ⁇ hism in question has already been characterized.
  • the first type of analysis is sometimes referred to as de novo characterization. This analysis compares target sequences in different individuals to identify points of variation, i.e., polymo ⁇ hic sites.
  • the second type of analysis involves determining which form(s) of a characterized polymo ⁇ hism are present in individuals under test.
  • suitable procedures for determining polymo ⁇ hic forms and thus polymo ⁇ hic profiles including, for example, the methods that follow. 1. Allele-Specific Hybridization (ASH)
  • ASH technology is based on the stable annealing of a short, single- stranded, oligonucleotide probe to a completely complementary single-strand target nucleic acid. Hybridization is detected from a radioactive or non-radioactive label on the probe. For each polymo ⁇ hism, two or more different probes are designed to have identical DNA sequences, except at the polymo ⁇ hic nucleotides. Each probe has exact homology with one allele sequence so that the complement of probes can distinguish all the alternative allele sequences. With appropriate probe design and stringency conditions, a single-base mismatch between the probe and target DNA prevents hybridization.
  • restriction fragment length polymo ⁇ hism refers to inherited differences in restriction enzyme sites (for example, caused by base changes in the target site), or additions or deletions in the region flanked by the restriction enzyme site that result in differences in the lengths of the fragments produced by cleavage with a relevant restriction enzyme.
  • a point mutation leads to either longer fragments if the mutation is within the restriction site or shorter fragments if the mutation creates a restriction site. Additions and transposable elements lead to longer fragments and deletions lead to shorter fragments.
  • An RFLP can be used as a genetic marker in the determination of segregation of alleles with quantitative phenotypes.
  • the restriction fragments are linked to specific phenotypic traits. More specifically, the presence of a particular restriction fragment can be used to predict the prevalence of a specific phenotypic trait. 3.
  • Polymo ⁇ hisms can be analyzed directly using the traditional dideoxy- chain termination method or the Maxam -Gilbert method (see Sambrook, et al, MOLECULAR CLONING, A LABORATORY MANUAL (2nd Ed., CSHP, New York 1989); and Zyskind et al, RECOMBINANT DNA LABORATORY MANUAL, (Acad. Press, 1988)).
  • Other nucleic acid sequencing methods including, but not limited to, fluorescence-based techniques (U.S. Patent No. 5,171,534), mass spectroscopy (U. S. Patent No. 5,174,962) and capillary electrophoresis (U.S. Patent No. 5,728,282) can also be used.
  • Drag-Tagging Oligonucleotides for Electrophoresis Oligonucleotides having additional chemical moieties that cause differential mobilities in an electrophoretic separation system can be used in analyzing polymo ⁇ hisms.
  • the addition of a molecular species increases the apparent molecular weight of a piece of amplified DNA, even DNA having only a single nucleotide polymo ⁇ hism present.
  • the added species, or drag tags can be attached covalently or non-covalently to the nucleic acid, either before or after labeling (for visualization of the electrophoretic band). Any charge associated with the added species is blocked or neutralized so that nucleic acid mobility remains dependent on size and not charge.
  • any of a number of different moieties can be attached to a nucleic acid to form a plurality of different sized amplification products.
  • moieties include, but are not limited to, phosphate monomers, acrylamide and polypeptides.
  • phosphate monomer units can be attached to each nucleotide monomer that is to be used in the PCR reaction. For example, assume one phosphate monomer is added to dATP; two phosphate monomers are added to dCTP; three phosphate monomers are added to dGTP; and four phosphate monomers are added to dTTP.
  • the resulting amplified polymo ⁇ hic nucleic acids contain different amounts of phosphate monomers depending on the nucleotide content. Hence, while the amplified products have the same numbers of base pairs, the different polymo ⁇ hic forms nonetheless may be size separated on an electrophoretic gel due to differences in phosphate monomer content.
  • the monomer units are added after amplification to specific nucleotides or to non-amplified nucleic acids prior to separation on the basis of size (e.g., by capillary electrophoresis).
  • Isozymes are a group of enzymes that catalyze the same reaction but vary in physical properties resulting from differences in amino acid sequence (and hence nucleic acid sequence). Some isozymes are multimeric enzymes containing slightly different subunits. Other isozymes are either multimeric or monomeric but have been cleaved from the proenzyme at different sites in the amino acid sequence. Nucleic acid variation of isozymes can be determined by hybridizing primers that flank a variable portion of an isozyme nucleic acid sequence to target nucleic acids contained in a sample obtained from an organism. The variable region is amplified and sequenced. From the sequence, the different isozymes are determined and linked to phenotypic characteristics.
  • Amplified variable sequences of the genome and complementary nucleic acid probes also can be used as polymo ⁇ hic markers.
  • amplified variable sequences refers to amplified sequences of the genome that exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. The presence of a specific variable sequence can be used to predict phenotypic traits.
  • a variable sequence of DNA can be amplified (e.g., utilizing the amplification techniques listed above) by template-dependent extension of primers that hybridize to flanking regions of the DNA obtained from a subject. The amplified products can then be sequenced. 7. Allele-Specific Primers and Hybridization
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymo ⁇ hism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity.
  • This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers and produces a detectable amplified product that can be characterized for the particular allelic form present in a nucleic acid sample. See, e.g., Gibbs, Nucleic Acid Res. 17:2427-2448 (1989) and WO 93/22456. 8. Single-Strand Conformation Polymo ⁇ hism Analysis
  • Alleles of target sequences can be differentiated using single-strand conformation polymo ⁇ hism analysis, which identifies base differences by alteration in electrophoretic migration of single-stranded PCR products, (see, e.g., Orita, et al, Proc. Nat'lAcad. Sci. USA 86:2766-2770 (1989).
  • amplified PCR products are denatured (e.g., according to known chemical or thermal methods) to form single- stranded amplification products that can refold or form secondary structures, depending in part upon the base sequence of the product.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of target sequences.
  • Polymo ⁇ hisms can also be identified by self-sustained sequence replication.
  • target nucleic acid sequences are amplified (replicated) exponentially in vitro under isothermal conditions using three enzymatic activities involved in retro viral replication: (1) reverse transcriptase, (2) RNase H, and (3) a DNA-dependent RNA polymerase (Guatelli, et al, Proc. Natl. Acad. Sci. USA 87:1874 (1990)).
  • RNase H RNase H
  • a DNA- dependent RNA polymerase a DNA-dependent RNA polymerase
  • Arbitrary fragment length polymo ⁇ hisms can also be used as polymo ⁇ hisms (Vos, et al, Nucl. Acids Res. 23:4407 (1995)).
  • the phrase "arbitrary fragment length polymo ⁇ hism” refers to selected restriction fragments that are amplified before or after cleavage by a restriction endonuclease. The amplification step permits easier detection of specific restriction fragments as compared to determining the size of all restriction fragments and comparing the sizes to a known control.
  • AFLP allows the detection of a large number of polymo ⁇ hic markers (see, supra) and has been used for genetic mapping of plants (Becker, et al, Mol. Gen. Genet. 249:65 (1995); and Meksem, et al, Mol. Gen. Genet. 249:74 (1995)) and to distinguish among closely related bacterial species (Huys, et al, Int 'l J. Systematic Bacteriol. 46:572 (1996)).
  • SSR methods are based upon high levels of di-, tri- or tetra-nucleotide tandem repeats within a genome. Dinucleotide repeats have been reported to occur in the human genome as many as 50,000 times with n varying from 10 to 60 (Jacob, et al, Cell 67:213 (1991)). The dinucleotide repeats have also been found in higher plants (Condit & Hubbell, Genome 34:66 (1991)).
  • SSR data is generated by hybridizing primers to conserved regions of the genome that flank the SSR region.
  • the dinucleotide repeats between the primers are amplified by PCR.
  • the resulting amplified sequences are then electrophoresed to determine the size, and therefore the number, of di-, tri- and tetra-nucleotide repeats.
  • Amplification products generated using the polymerase chain reaction can be analyzed through the use of denaturing gradient gel electrophoresis. Different alleles are identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR TECHNOLOGY, PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION, (W.H. Freeman and Co, New York, 1992), Chapter 7. 13. Single base extension methods
  • Polymo ⁇ hisms can also be detected by single base extension.
  • a primer is designed to hybridize to a target sequence so that the 3' end of the primer immediately abuts but does not overlap a polymo ⁇ hic site.
  • the target sequence is then contacted with primer and at least one nucleotide (typically labelled), that is complementary to the base occupying the polymo ⁇ hic site in one allelic form. If that allelic form is present, then the primer is extended and becomes labelled.
  • biallelic polymo ⁇ hic sites are analyzed by including two differentially labelled dideoxynucleotides respectively complementary to bases occupying the polymo ⁇ hic site in first and second allelic forms of the target. Analysis of label present in the extended primer indicates whether one or both of the allelic forms are present in a target sample.
  • high throughput screening involves providing a library of polymo ⁇ hic forms of DNA including RFLPs, AFLPs, isozymes, specific alleles and variable sequences, including SSR. Such "libraries" are then screened against genomic DNA from the subjects in the treatment study. Once the polymo ⁇ hic alleles of a subject have been identified, a link between the polymo ⁇ hic DNA and the treatment effect can be determined through statistical associations.
  • hybridization can be performed in many different formats. For example, for those methods involving hybridization reactions, hybridization can be performed in a 96-, 324-, or a 1024-well format or in a matrix on a silicon chip.
  • a dot blot apparatus is used to deposit samples of fragmented and denatured genomic DNA on a nylon or nitrocellulose membrane. After cross-linking the nucleic acid to the membrane, either through exposure to ultra-violet light if nylon membranes are used or by heat if nitrocellulose is used, the membrane is incubated with a labeled hybridization probe. The membranes are washed extensively to remove non- hybridized probes and the presence of the label on the probe is determined.
  • the labels are inco ⁇ orated into the nucleic acid probes by any of a number of methods well known to those of skill in the art.
  • a label is simultaneously inco ⁇ orated during the amplification procedure in the preparation of the nucleic acid probes.
  • PCR polymerase chain reaction
  • labeled primers or labeled nucleotides provide labeled amplification product.
  • transcription amplification using a labeled nucleotide e.g., fluorescein- labeled UTP and/or CTP
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,
  • radiolabels are detected using photographic film or scintillation counters and fluorescent markers are detected using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • robotic systems have been developed for high throughput screening, particularly in a 96 well format. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD.
  • Polymo ⁇ hic forms of DNA can also be identified by hybridization to nucleic acid arrays, some examples of which are described by WO 95/11995 (inco ⁇ orated by reference in its entirety for all pu ⁇ oses).
  • solid phase arrays are adapted for the rapid and specific detection of multiple polymo ⁇ hic nucleic acids.
  • a nucleic acid probe is linked to a solid support and a target nucleic acid is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore. If the target is labeled, hybridization is detected by detecting bound fluorescence. If the probe is labeled, hybridization is typically detected by quenching of the label by the bound nucleic acid. If both the probe and the target are labeled, detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels.
  • probe design is influenced by the intended application. For example, where several probe-target interactions are to be detected in a single assay, e.g., on a single DNA chip, it is desirable to have similar melting temperatures for all of the probes. Accordingly, the lengths of the probes are adjusted so that the melting temperatures for all of the probes on the array are closely similar (different lengths for different probes may be needed to achieve a particular Tm where different probes have different GC contents). Although melting temperature is a primary consideration in probe design, other factors are optionally used to further adjust probe construction.
  • capillary electrophoresis can be used to analyze polymo ⁇ hism.
  • size based separations e.g., RFLP and SSR
  • capillary electrophoresis tubes are filled with the separation matrix.
  • the separation matrix contains hydroxyethyl cellulose, urea and optionally formamide.
  • the RFLP or SSR samples are loaded onto the capillary tube and electrophoresed. Because of the small amount of sample and separation matrix required by capillary electrophoresis, the run times are very short.
  • Electrophoresis can also be performed in microchannel plates. These plates have channels less than 100 ⁇ in diameter etched on a solid substrate. By virtue of their smaller dimensions, they allow for even faster separations of nucleic acids in a separation matrix. Using these etched plates, samples can be evaluated with a high throughput format. In another high throughput format, multiple capillary tubes are placed in a capillary electrophoresis apparatus. Samples are loaded onto the tubes and electrophoresis of the samples is run simultaneously. See, for example, Mathies & Huang, Nature 359:167 (1992). Because the separation matrix is of low viscosity, after each run, the capillary tubes can be emptied and reused.
  • the invention can be illustrated by the example of studying serum cholesterol and the effects drugs may have on this biological condition. It has been established that up to 80% of the variance in serum cholesterol can be attributed to genetics (see, for example, R. A. King, J. I. Rotter & A. G. Motulsky, THE GENETICS BASIS OF COMMON DISEASE, Oxford University Press, 1992).
  • the power of genetic matching can be realized in two other ways. For example, using the same number of patients (50) in each arm, with genetic matching the difference that can be resolved with the same power drops from 20 to 8.8. Likewise, the power of the study increases from .8 to greater than 0.99 assuming genetic matching and a difference of 20. If genetic matching cannot be fully achieved, some degree of matching can still have a favorable impact on such studies. This is illustrated in Table 1 below. TABLE 1
  • the following example is illustrative of the method of identifying underlying genetic factors that influence the response to treatment and the use of this information in the design of clinical trials.
  • the response of treated individuals from the first sub-population, A has mean ⁇ A and variance, ⁇ A .
  • the mean and variance of response are given by ⁇ B and ⁇ , respectively. No assumption is made about the shape of either distribution (i.e. they do not have to be normal).
  • the mean and variance of response is given by ( ⁇ A ⁇ A) an &
  • both the treated (case) and untreated (control) populations include a mixture of individuals sampled from the two distributions.
  • N is the total population size and ⁇ x ⁇ ,X2,—,X j ,...x js/ ) is a set of random variables, each describing an individual in the sample, the expectation of the mean response in the sample is given by,
  • the variance of the mean response in such a case depends on both the variances of each of the two distributions (A and B) and on the difference between the means of these distributions. This variance can be expressed in terms of the sum of the variances of the individuals,
  • V[x t ] E[V[x ⁇
  • V[x] Min[ ⁇ A I N, ⁇ B I N] . That is, the variance when the sub-populations are ignored is always larger than the variance of one of the sub-populations. This can intuitively be seen from the shape of equation 6.
  • the variance is the weighted sum of the two population specific variances ( ⁇ A 2 , ⁇ B 1 ) plus a term representing the difference between the two population means ( ⁇ - ⁇ ) .
  • the variance consists of both the within population variance and the between population variance.
  • Theorem and has mean and va ⁇ ance (p ⁇ A + q ⁇ B , a — — — ) .
  • all individuals from the sub-population are genotyped for k equally informative markers. This is sometimes the case when markers are chosen at random (i.e. if nothing is known about genes involved in responsiveness). Additional markers will usually provide decreasing information (i.e. though the k + 1 th marker increases the probability of correctly assigning an individual to a sub-population, it provides less information than the k th marker); this does not necessarily have to be the case but often is the case. For example, if there is a priori knowledge of the genes involved in response, these are typically examined first.
  • the genetic matching is based on relatedness (i.e., the overall degree of genetic similarity in the genome) and hence the first few markers will be highly informative with diminishing information from each additional markers.
  • sample mean is given by ( ⁇ A , ⁇ A 2 l N) for population A and ( ⁇ B , ⁇ 2 I N) for population B (using the information to set p - 0 and so select non-responders) as expected.
  • C Power of the clinical trial with genetic matching.
  • the two samples are selected to be of equal size, but this does not have to be so.
  • N is reasonably large (>30)
  • standard theory of normal distributions can be used to show that the necessary sample size to detect a difference in response between the treated and placebo groups at the a% level with power ⁇ is,
  • Table 2 gives the number of markers ( k ), the probability of the selected individual coming from group A (i.e., being correctly identified as a responder) ( p ), the variance of the sample mean for the treated population ( V[x] ) and the sample size required in each arm of the trial ( N ).
  • the within population variance is fixed at 64 for both genetic subpopulations and in both treated and untreated samples.
  • Table 2 shows that, when no markers are genotyped, the variance is 70. This increase in the variance is entirely due to the difference in the response due to the underlying genotype. When this is accounted for (when p -» 1 ), the variance returns to the expected value of 64. This inflation in variance has a marked effect on the necessary sample size (note in equation 11 , the sample size does not increase linearly increasing variance, but rather with the square of the variance). Where 83 individuals are needed in each arm of the trial if no genetic information is available, only 20 individuals are needed if individuals can be co ⁇ ectly assigned as responders using their polymo ⁇ hic profile.
  • the two genetic sub-populations A and B there are two genetic sub-populations A and B, and a single bi-allelic SNP (single nucleotide polymo ⁇ hism) that is present in both sub- populations.
  • the population-wide frequency of the rare allele i.e., the allele of the pair that has the lower frequency in the general population
  • p(g) (p A + p B ) 12.
  • the allele g is associated with increased response to the treatment.
  • the increased response can be due to the function of the allele itself, but more usually arises due to the allele being in linkage disequilibrium with some (unknown) genetic factor or mutation.
  • an SNP lies in a region that is known to harbor a gene involved in response to treatment and sub-population A consists of high responders whereas sub-population B consists of low responders.
  • conditional probabilities can also be expressed (using Bayes' theorem) as,
  • the strength of the association is encapsulated in the difference between the allele frequencies in the sub-populations.
  • G € ⁇ g, ,...,g k ⁇ represents the genotype of an individual at a set of bi-allelic markers with frequencies ⁇ p x ,...,p k ⁇ in sub-population A and ⁇ q x ,...,q k ⁇ in sub-population B. Then, by B ayes' Theorem,
  • Table 3 shows that the probability of co ⁇ ectly assigning an individual to the correct sub-population increases rapidly with the number of markers genotyped.
  • the markers are randomly chosen (not known to be associated with a specific gene) and the difference in the frequency of the rare allele between the two sub- populations is chosen to be 0.1.
  • Information of the type given in Table 3 can be used directly to categorize individuals into sub-populations or as a criterion for enrollment into a trial.
  • individuals having the rare allele at 1 or 0 of the 5 polymo ⁇ hisms can be included in a clinical trial. This increases the probability of the trial containing individuals that will respond to the treatment (where it is assumed that sub-population A responds to treatment and sub-population B does not).
  • the probability of belonging to sub-population A is 0.713 and for individuals with 1 rare allele, the probability of belonging to sub-population A is 0.620.
  • this selection criterion greatly increases the proportion of individuals from sub-population A enrolled in the clinical trial.
  • This simple method of selection is only illustrative and many other more complex procedures (for example, cluster analysis) can be employed depending on the number of polymo ⁇ hisms and their respective allele frequencies.
  • the two alleles are at similar frequency
  • a set of markers, K in number are genotyped in a sample of 2000 patients who are known to belong either to sub-population A or sub-population B (from a previous clinical trial).
  • Table 4 gives the probability of assigning an individual to the correct sub- population for 2, 5, 10, 20 and 50 markers. Values are given for both types of markers and for a mixture of the two. In this example, all markers are assumed to be independent of one another. If this were not the case, other, more powerful, statistical methods can be applied (for example, methods of classification trees (Breiman et al., Classification and Regression Trees, CRC Press, 1984).
  • markers for which the two alleles have similar frequencies are more effective in determining which group and individual belongs to, than are markers with very different allele frequencies.
  • An equal mixture of markers from these two classes provides, as expected, intermediate results.
  • markers for which both alleles are common perform better than those for which one allele is very rare.
  • the present invention has a number of uses with regard to treatment studies generally, and clinical trials in particular.
  • the invention includes the use of a polymo ⁇ hic profile to conduct a clinical trial on a population of patients having the same disease, wherein the polymo ⁇ hic profile includes at least one polymo ⁇ hic site not known to be associated with the disease.
  • the invention also includes the use of a polymo ⁇ hic profile to conduct a reanalysis of data from a clinical trial in which statistical significance is determined on subpopulations of original treated and control groups selected for similarity of polymo ⁇ hic profile.
  • the invention also includes the use of a polymo ⁇ hic profile to divide a population of individuals subject to a clinical trial into a plurality of subsets, the members of a subset showing greater similarity of polymo ⁇ hic profile to each other than members in different subsets.

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Abstract

L'invention concerne des procédés, des programmes informatiques et des systèmes informatisés pouvant servir à évaluer l'efficacité de divers types de procédures de traitement (p.ex., d'essais cliniques) en fonction du génotype d'un sujet. Grâce à l'appariement génétique des groupes traité et de contrôle, les procédés et systèmes de l'invention réduisent la variance globale de l'étude et nécessitent ainsi moins de sujets pour mener des essais visant à examiner l'efficacité ou l'effet des procédures de traitement, et ce avec un degré de certitude plus élevé et/ou une précision ou une capacité de discrimination plus grande. Certains procédés de l'invention consistent à sélectionner des sous-populations traitées et de contrôle à partir des populations traitée et de contrôle sur la base des similitudes du profil polymorphe, les populations traitée et de contrôle ayant été traitées selon des procédures de traitement et de contrôle, respectivement. On détermine ensuite s'il existe entre les sous-populations traitées et de contrôle une différence statistiquement signifiante d'un paramètre test servant d'estimation de la procédure de test.
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