WO2021026293A1 - Compositions and methods relating to identification of genetic variants - Google Patents

Abstract

The invention provides compositions and methods for detecting a particular genetic polymorphism in a complex genomic environment. The present disclosure provides compositions and methods for genotyping a genetic variant located in a complex genomic environment, such as a genetic locus having multiple structural isoforms of a gene. The key advantages of the claimed methods are their reliance on the amplification of short DNA fragments involving only two sets of primers and do not require extensive sequencing step(s).

Description

COMPOSITIONS AND METHODS RELATING TO IDENTIFICATION OF

GENETIC VARIANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 62/883,562, filed on August 6, 2019, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to new methods for detecting a genetic variant in a complex genetic background and methods for treating patients.

BACKGROUND

[0003] Carboxylesterases are a multi-gene family of enzymes that catalyze the hydrolysis of esters, amides, thioesters, and carbamates. Mammalian carboxylesterases (CESs) have prominent roles in the hydrolysis of numerous and diverse compounds including carboxylic acid esters, carbamates, thioesters, and amide containing agents. These substrates are represented in almost every drug class, as well as in many prodmgs and agents purposely formulated as esters for improving oral bioavailability of the active moiety.

[0004] Human carboxylesterase 1 (hCESl) is the major carboxylesterase expressed in the human liver. hCESl contributes about 80% of total hepatic hydrolytic activity (Imai T et al., Drug Metab Phamacokinet 2006; 23:73-84). hCESl is responsible for metabolizing numerous important therapeutic drugs, and for the hydrolysis of a variety of ester- and amide-containing drugs and prodrugs, such as the antiplatelet prodrug, clopidogrel; the antiviral, oseltamivir; attention deficit hyperactivity disorder (ADHD) medications such as methylphenidate; angiotensin converting enzyme (ACE) inhibitors including imidapril, enalapril,trandolapril, ramipril, and temocapril, and anticancer drugs such as capeciain. hCES 1 is also involved in the detoxification or metabolic activation of various other drugs (Lewis JP et al., Pharmacogenet Genomics 2013; 23(1): 1-8).

[0005] hCES 1 is encoded by the CES 1 gene family which contains four structural genomic isoforms, CES1 and its variant, CESlvar, and a pseudogene, CES1P1 and its variant, CESlPlvar. CES1 and CESlvar are allelic genes whose DNA sequences are highly homologous, differing only in the 5’ untranslated region (5’UTR) and exon 1. The pseudogene CES1P1 and its variant are located on chromosome 16q in tail-to-tail configuration with the allelic genes. The combinations of these four structural genomic isoforms leads to four CES1 haplotypes designated A, B, C and D, and represent the following combinations of the allelic CES1 genes and pseudogenes: A (CES1P1-CES1),

B (CES lPlvar-CES 1), C (CESlPl-CESlvar) and D (CESlPlvar-CESlvar). See Sanford JC et al, Pharmacogenet Genomics 2016; 26:197-207. The combinations of CES1 haplotypes A, B, C and D make up 10 diplotypes (A/A, A/B, A/C, A/D, B/B, B/C, B/D, C/C, C/D and D/D), any one of which will be found in a particular individual.

[0006] The single nucleotide polymorphism (SNP) designated rs71647871 identifies a 428G>A single nucleotide variation in exon 4 of CES1. Each of the four structural variants of CES 1 has an identical nucleotide sequence in the immediate vicinity of this SNP. This means that methods which identify only the SNP itself, without more, will fail to identify whether the SNP occurs within one of the CES1 allelic genes or within one of the pseudogenes. This is important because its occurrence within one of the allelic genes would have a much greater impact on drug metabolism. The minor, variant allele (A) of rs71647871 results in an amino acid change of glycine to glutamic acid at positionl43 (143G>E). This mutation reduces the hydrolytic activity of the CES1 protein to the point of effectively abolishing its metabolic activity. One clinical study demonstrated that the CES1 143G>E variant increased the antiplatelet effects of clopidogrel by reducing its hydrolysis. Based on this, the CES1 143G>E variant has been proposed as a biomarker of clopidogrel response (Lewis JP et al., Pharmacogenet Genomics 2013; 23(1): 1-8). But given the importance of CES1 in the metabolism of many different drugs, carriers of this SNP in one or both of the allelic CES 1 genes are likely to show clinically significant variations in drug metabolism more generally. Although the minor allele frequency of rs71647871 is relatively rare (about 3.7%, 4.3%, 2.0% and 0% in European- American, African-American, Hispanic, and Asian ethnic populations, respectively), its significant effect on CES 1 enzymatic activity, and consequently on the metabolism of a wide variety of therapeutic drugs, makes its accurate identification an important clinical tool for ensuring that patients receive the most appropriate medication at an appropriate dose. See e.g., Zhu HJ et al, Pharmacogenet Genomics 2012; 22:215-218.

[0007] The accurate genotyping of this variant is difficult due to the presence of multiple CES1 haplotypes, each having highly conserved sequences overall, as well as in the vicinity of rs71647871, as discussed above. The very high DNA sequence similarity between the CES 1 isoforms and combination haplotypes makes it difficult to identify whether the variant occurs within an allelic gene or a pseudogene using routine genotyping methods such as allele- specific PCR and real-time PCR. There have been several reports using these methods for genotyping rs71647871. See Sanford JC el al, Pharmacogenet Genomics 2016, 26:197-207; Lewis JP et al., Pharmacogenet Genomics 2013; 23(1): 1-8; Wang X et al., Pharmacogenomics J. 2015; 16(3):220-30; Genetic Testing and Molecular Biomarkers 2010 14:749-751. Some of these reports have indicated that a thirty percent reduction in mRNA expression did not detectably alter protein expression or metabolizing activity ( See Sanford JC et al, Pharmacogenet Genomics 2016, 26:197-207). Sanford et al teaches CeslVar genotypes have no impact on the metabolism of clopidogrel, enalapril or methylphenidate. A significant shortcoming of these methods is their inability to differentiate heterozygous from homozygous carriers in the allelic CES1 genes. Instead, because these methods do not identify where the variant occurs, whether it occurs in the pseudogene or the allelic gene, interpretation of the results is prone to error and leads to false heterozygous characterizations and masking of homozygous mutants. Usually, short-range PCR methods are based on the amplification of the regions in close proximity to the variant. Since these regions are highly homologous between the different CES1 isoforms, all isoforms, including pseudogenes will be amplified. This prevents the identification of the homozygous carriers, since even if both active alleles contain the variant, the result appears heterozygous because of the presence of the wild type SNP in the pseudogenes.

[0008] Long-range PCR combined with direct sequencing ( e.g ., using the Sanger method) has also been used to genotype rs71647871. Zhu et al, Pharmacogenet. Genomics 2012 March; 22:215-218; see also Markowitz and Zhu US 2014/0162896. In this method, two sets of primers were designed to take advantage of differences in the promoter regions between the allelic CES 1 genes and the pseudogenes in order to specifically amplify separate fragments from the allelic and pseudogenes. Due to the reliance on differences in the promoter regions of the genes, the resulting PCR products were large, on the order of about 14 kilobase-pairs (kb), and spanning exon 1 to exon 6 of CES 1. These long-range PCR products were then sequenced by direct sequencing or PCR-based sequencing to identify the status of the rs71647871 variant. Although this method was able to detect the variant status of rs71647871 in the allelic CES1 genes, its reliance on long-range PCR, with a reaction time of about 13 to 14 hours, makes it less than ideal for clinical use. The lengthy reaction time slows results and delays care.

Further long-range PCR utilizes more reagents, may require alternative polymerases and may have reduced fidelity rates as compared to typical PCR reactions.

[0009] Wang et al Pharmacogenomics J. 2016 June; 16(3): 220-230 recognized the challenges associated with determining the CESl/CESlvar/CESlPl/CESlPlVar genotypes due to the high similarity of the CES1 and CESlpl genes. Wang et al concluded CESl/CESlVar genotypes were not associated with the variability in CES lexpression and activity in human livers.

[0010] There is a need for improved methods for genotyping SNP variants that can accurately identify carriers and differentiate heterozygous from homozygous carriers of the variant allele, particularly where those variants occur in a genetic locus characterized by multiple structural isoforms of a gene having high sequence identity within the region where the variant occurs. The present disclosure addresses this need.

SUMMARY OF THE INVENTION

[0011] The present disclosure provides compositions and methods for genotyping a genetic variant located in a complex genomic environment, such as a genetic locus having multiple structural isoforms of a gene. The key advantages of the claimed methods are their reliance on the amplification of short DNA fragments involving only two sets of primers and do not require extensive sequencing step(s). The streamlined simplicity allows the methods to be fast, inexpensive and applicable for high-throughput analysis. The current methods require fewer reagents and resources as compared to time consuming long-range PCR methods or methods requiring Sanger based sequencing.

[0012] In embodiments, the disclosure provides a method for genotyping a single nucleotide polymorphism (SNP) in a biological sample from a subject, the SNP occurring within a genetic locus of a gene having multiple structural isoforms, the SNP consisting of a major allele and a minor allele, the method comprising: amplifying a first DNA fragment from the biological sample using a polymerase chain reaction (PCR) and a first pair of primers designed to amplify the first DNA fragment, the first DNA fragment consisting of a portion of the gene which contains the SNP and which is the same in each of the multiple structural isoforms of the gene, to produce a first PCR reaction product, PCR A; amplifying a second DNA fragment from the biological sample using PCR and a second pair of primers designed to amplify the second DNA fragment, the second DNA fragment consisting of a portion of the gene unique to subset of the multiple structural isoforms, to produce a second PCR reaction product, PCR B; genotyping the SNP in each of the first and second PCR reaction products, PCR A and PCR B ; and comparing the relative amounts of the variant allele in each of PCR A and PCR B, thereby determining the status of the SNP within the subset of the multiple structural isoforms.

[0013] In embodiments, the disclosure also provides a method for genotyping the rs71647871 SNP in a biological sample from a subject and identifying its status within the four structural isoforms of the human CES 1 genetic locus, said locus consisting of two allelic isoforms, CES1 and CESlvar, and two non-allelic isoforms, CES IP and CESlPvar, the SNP consisting of a major allele (G) and a minor allele (A), the method comprising: amplifying a first DNA fragment from the biological sample using a polymerase chain reaction (PCR) and a first pair of primers designed to amplify the first DNA fragment, the first DNA fragment consisting of a portion of the CES 1 gene which contains the rs71647871 SNP and which is the same in each of the four structural isoforms of the gene, to produce a first PCR reaction product, PCR A; amplifying a second DNA fragment from the biological sample using PCR and a second pair of primers designed to amplify the second DNA fragment, the second DNA fragment consisting of a portion of the CES 1 gene unique to the allelic isoforms of the CES 1 gene, to produce a second PCR reaction product, PCR B ; genotyping the rs71647871 SNP in each of the first and second PCR reaction products, PCR A and PCR B; and comparing the relative amounts of the variant allele in each of PCR A and PCR B, thereby determining the status of the rs71647871 SNP within the allelic isoforms of the CES1 gene.

[0014] In embodiments, the genotyping of the SNP is performed by a high throughput method. In embodiments, the high throughput method comprises mass spectrophotometry or flow cytometry, or a combination of the two. In embodiments, the mass spectrophotometry comprises matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF). In embodiments, the method comprises a primer extension step. In embodiments, the method comprises incorporating a labelled deoxyribonucleotide (dNTP) specific to the minor or major allele during a primer extension step and detecting the labelled dNTP in each of the extension PCR products A and B .

[0015] In embodiments, the biological sample is a buccal swab.

[0016] In embodiments, the disclosure provides a kit of parts comprising at least two sets of PCR primers, each adapted to selectively amplify a common fragment of DNA from (1) each of the multiple structural isoforms of the gene, for example, each of the four structural isoforms of the CES1 gene; and (2) a subset of the structural isoforms of the gene, for example, the allelic isoforms of the CES1 gene. In each of (1) and (2), the DNA fragment contains the genetic variant of interest, for example, the rs71647871 SNP of the CES1 gene.

[0017] In embodiments, the PCR primers are modified. In embodiments, the PCR primers are chemically modified. In embodiments, the PCR primers are modified to contain a detectable label. In embodiments the PCR primers have different masses. In various embodiments the detectable label is fluorescent. Methods may include, but are not limited to Luminex and Maldi-TOF

[0018] In embodiments, the PCR primers of the first primer pair are selected from the group of primers that are 15-30 nucleotides long, have compatible melting temperatures (Tm) and amplify CES1, CES1VAR, CES1P1 and CES1P1VAR. The primers may further comprise method- specific tags that are known in the art; such method- specific tags may increase the primer length.

[0019] In embodiments the primers of the second pair of primers are selected from the group of primers comprising primers that are 15-30 nucleotides long, have compatible Tm and amplify a unique identifying region from a subset of CES1, CES1VAR, CES1P1 and CES1P1VAR. The primers may further comprise method- specific tags that are known in the art; such method- specific tags may increase the primer length. By a subset of multiple structural isoforms is intended the total number (n) of structural isoforms minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to n-1 isoforms. In an embodiment the total number of structural isoforms is 4 and by subset is intended 1, 2 or 3 isoforms. In an embodiment the structural isoforms are CES1, CES1VAR, CES1P1 and CES1P1VAR and a subset of the structural isoforms includes 1, 2 or 3 of the isoforms.

[0020] In embodiments, the kit further contains one or more additional reagents selected from the group consisting of a buffer, a detection reagent, a polymerase enzyme, a positive control sequence, and a negative control sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figs. 1A and IB: Mass spectrum results of CES1 rs71647871 status. The extension products of the G (wild type) or A (variant) allele from the PCR DNA fragments containing rs71647871 were separated by absolute mass on MALDI-TOF.

PCR A contains an amplified fragment of exon 4 from each of the four CES1 genes. PCR B contains an amplified fragment of exon 4 from only the allelic genes, CES 1 and CESlvar. Wild type, G (G/G); heterozygote, G>A (G/A); homozygote, A (A/A) at the SNP rs71647871. Fig. 1A: Mass spectrum of rs71647871 status in CES1 using a MassARRAY iPLEX™ genotyping platform without mutation in interfering SNPs. Fig. IB: Mass spectrum of rs71647871 status in CES1 using a MassARRAY iPLEX™ genotyping platform with mutation in interfering SNPs.

[0022] Fig. 2: Luminex xTAG genotyping of SNP rs71647871 of CES1. Cluster maps from two sets of samples for genotyping of CES1 rs71647871. The extension products of the G (wild type) or A (mutant) from PCR DNA fragments containing rs71647871 were amplified with ASPE through Luminex Universal Tag sorting system. The genotype was determined by Median florescence intensity (MFI) by absolute MFI > 1000. PCR A and PCR B are as defined in Fig. 1 above.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides methods for accurately genotyping a genetic variant of interest located within a complex genetic locus, such as one characterized by multiple structural isoforms of a gene, each isoform having high sequence identity within the region where the variant occurs. The methods described here are able to discriminate the status of the variant itself, for example whether the major allele is present on both alleles of the DNA (wild-type status), or on only one allele (heterozygous status), or whether the minor allele is present on both alleles of the DNA (homozygous variant status). In addition, the methods described here are able to determine the status of the variant within its genomic context, that is, where it is located within the one or more structural isoforms of the gene. In embodiments, the genomic context refers to whether the variant is located in an allelic or non-allelic isoform of the gene.

[0024] The present methods provide for more accurate and detailed identification of genetic variants that are difficult to accurately identify using conventional genotyping methods, such as those occurring within genetic loci characterized by multiple structural isoforms of a gene. Different structural isoforms of a gene may include multiple allelic and non-allelic isoforms which may differ in their level of expression of mRNA and/or protein, either globally or within a particular type of tissue, or in their ability to express functional protein at all. The presence of such multiple allelic and non-allelic isoforms generates multiple haplotypes for the gene which in turn account for multiple possible diplotypes, any one of which is carried by an individual. The occurrence of a genetic variant which impacts the expression or activity of the protein encoded by the gene may have different clinical consequences depending on which structural isoform of the gene it occurs in. Thus, for example, it may be important to identify whether the genetic variant occurs within an allelic isoform or within a pseudogene.

[0025] Where a genetic variant within the gene results in a mutation affecting protein expression and/or function, the ability to identify the location of the genetic variant within a particular haplotype may have important consequences for predicting the biological effects of the variant. This is the case with the CES1 gene, as discussed above in the background section. For example, the presence of two allelic and two non-allelic isoforms of CES1 results in four CES1 haplotypes (A, B, C, and D) and ten diplotypes, any one of which may be present in an individual. The rare SNP rs71647871 (428G>A) results in an amino acid mutation at position 143 which changes a glycine residue to glutamic acid, effectively inactivating the protein. The region of the gene containing this SNP is the same in each of the four CES1 isoforms. However, the pseudogenes are expressed at low levels, and one does not result in protein expression at all, so the biological consequences of carrying the minor allele of rs71647871 are highly dependent on whether it occurs within one of the CES1 pseudogenes or within one of the allelic genes. For example, the presence of the inactivating mutation would be of less biological significance if it occurs in one of the non-allelic isoforms because each either fails to encode a functional protein (CES1P1) or encodes a functional protein (CESlPlvar) that is nevertheless expressed at much lower levels in the liver than either of the CES1 allelic isoforms. Accordingly, it can be of critical importance to identify whether an individual is a heterozygous or homozygous carrier of the minor allele of the rs71647871 in CES1 or CESlvar, as distinguished from carrying this inactivating mutation in either CES1P1 or CESlPlvar. As discussed above, since the CES1 and CESlPlvar isoforms encode almost the same functional mature protein and share highly homologous DNA sequences, prior art methods have not been able to distinguish the homozygous minor allele of this SNP in the allelic genes from heterozygous. The methods described here allow for the identification of the status of rs71647871 (428G>A) in the context of the allelic and non allelic isoforms of CES1. The status of this SNP is selected from homozygous wild-type, also referred to as the ‘major allele’ of the SNP because it is the most common genotype, G/G; heterozygous (G/A), and homozygous mutant or variant, also referred to as the ‘minor’ allele of the SNP because it is the least common genotype, A/A. A patient or subject with reduced CES1 activity would be considered a CES1 poor metabolizer. The presence of the 428G>A SNP in the CES1 or CESlvar gene significantly reduces activity of the CES1 gene product. CES1P1 does not encode a functional protein, and CESlPlvar encodes a functional protein expressed at much lower levels than either of the CES1 isoforms and therefore presence of the 428G>A SNP in CES1P1 or CESlplvar will not have a significant effect on overall activity. “CES1 poor metabolizer genotypes” are those which are homozygous for the 428G>A SNP in the CES1 or CESlvar gene.

[0026] An SNP is a particular type of genetic variant characterized by a single nucleotide difference occurring in genomic DNA among individuals of a species, typically at a frequency of at least 1% in the population. SNPs may fall within coding regions of genes ( e.g ., exons), within non-coding regions of genes ( e.g ., introns), in the 3’ or 5’ untranslated regions of genes, or in the intergenic regions between genes. Typically an SNP will occur in the genome as one of two specific nucleotides at a given location. The nucleotide that is most prevalent among members of the species will be denoted the “major” allele while the least prevalent will be the “minor” allele. The minor allele is also sometimes referred to as the variant or mutant allele, to distinguish from the major allele which may also be referred to as the wild type allele.

[0027] The present disclosure exemplifies the application of the claimed methods to a genetic variant that is a single nucleotide polymorphism (SNP). However, other types of genetic variants may be genotyped in a similar manner. For example, other types of genetic variations include short deletion and insertion polymorphisms (indels or DIPs), short tandem repeats, and multinucleotide polymorphisms. A database of genetic variation is maintained by the National Center for Biotechnology Information and the National Human Genome Research Institute in the U.S. See Sherry et al. (2001). dbSNP: the NCBI database of genetic variation. Nucleic Acid Res., 29: 308-311. Unique genetic variants are assigned a reference number referred to as a reference SNP cluster number (rs#).

[0028] PCR amplification methods are known in the art. Any PCR amplification method used in the art is suitable for the claimed methods. However, it is recognized that PCR amplification methods that are quantifiable, that amplify sequences proportionally to the original sample set, that are bias -corrected or bias-minimized may be preferable. PCR amplification methods may include but are not limited to, RT-PCR, real time PCR, multiplex PCR, real time quantitative RT-PCR, nested PCR and PCR.

[0029] In embodiments, the methods described here utilize at least 2 sets of PCR primers designed to differentially amplify relatively short (from about 50 bp to about 4kbp, from about 50 bp to about 3.5 kbp, from about 50 bp to about 3 kbp, from about 50 bp to about 2.5 kbp, from about 50 bp to about 2 kbp, preferably from about 100 bp to 1.5 kbp, from about 100 bp to about 1.4 kbp, from about 100 bp to about 1.3 kbp from about 100 bp to about 1200 bp, from about 100 bp to about 1100 bp, from about 100 to about 1000 bp, from about 100 to about 900 bp; more preferably from about 100 to 800 bp, from about 100 to about 700 bp, from about 100 to about 600 bp, from about 100 to about 600 bp and from about 100 to about 550 bp PCR fragments from a region of a genetic locus of a gene characterized by multiple structural isoforms of the gene, the region containing a genetic variant of interest, such as an SNP. The at least 2 sets of primers produce at least two different PCR reaction products, PCR A and PCR B, representing two different sets of DNA fragments from the genetic locus. PCR A comprises DNA fragments containing the genetic variant of interest from each of the multiple structural isoforms of the gene. PCR B comprises DNA fragments containing the genetic variant of interest, but only from a subset of the multiple structural isoforms of the gene, for example, the allelic isoforms. Standard genotyping methods, preferably high throughput genotyping methods, are used to identify the status of the genetic variant of interest in each of the PCR reaction products. The method then utilizes a ratio of the amount of the genetic variant of interest in each of the PCR reaction products, e.g., PCR A and PCR B, in order to determine the genotype of the variant in the subset of structural isoforms of the gene, such as in the allelic isoforms.

[0030] In a particular embodiment, the methods utilize 2 sets of PCR primers designed to differentially amplify either (A) a fragment of exon 4 of the CES 1 gene containing the genetic variant rs71647871, the fragment being common to each of the four structural isoforms of CES1; or (B) a fragment of exon 4 of the CES1 gene that is common only to the allelic isoforms of CES1, where the fragment of exon 4 amplified in either PCR A or PCR B is from about 100 bp to about 1.5 kb, preferably from about 200 bp to 500 bp, or from about 200 bp to 300 bp. The small size of the amplified fragments in the two reactions, PCR A and PCR B, means that they can be rapidly and accurately genotyped at the genetic variant rs71647871 using high throughput methods for genotyping single nucleotide polymorphisms, including for example, methods utilizing mass spectrophotometry such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and microarray based nucleic acid detection technologies.

[0031] By “shared region” is intended a segment, length or region for which the DNA sequence is similar, indistinguishable, or highly conserved in each of the four structural isoforms of the gene. Although one or more shared regions may exist for the four structural isoforms of the gene, any single shared region will not include DNA sequence or sequence information that could identify the structural isoform which was the target source.

[0032] By unique region is intended a region of the allelic isoforms of the CES 1 gene that comprises a nucleotide sequence specific to one allelic isoform of the CES 1 gene such as but not limited to a unique identifying sequence. It is recognized that a PCR amplification production may comprise both a unique region and some non-unique regions such as but not limited to primer binding sites that allow amplification of unique regions from one or more isoforms using the same primers. The presence of a unique region allows identification of the isoform from which a PCR reaction product was obtained. A “unique identifying sequence” indicates the source isoform of the PCR product.

[0033] The methods described here involve detecting a genetic variant in a biological sample containing DNA from a subject, preferably a human subject, e.g., a patient. In embodiments, the biological sample contains a plurality of cells having diploid DNA content. In embodiments, the biological sample is a tissue sample that includes diploid cells of a tissue (epithelial cells) or organ (e.g., skin cells). Such cells or tissue may be obtained, for example, from a cheek swab, or from a biopsy. In embodiments, the biological sample is a fluid sample, for example, saliva, blood, blood serum, plasma, or urine. In embodiments, the biological sample is a buccal swab.

[0034] The methods described here may be useful in selecting a medication for a patient and/or in selecting or adjusting the dose of a medication for a patient. The methods may also be useful as part of a screen to assess a patient’s metabolic capacity, meaning the patient’s ability to metabolize medications via a known pathway, such as a CES 1 -dependent pathway; or in predicting a patient’ s responsiveness to a particular medication, for example a medication that is metabolized via a CES-1 dependent pathway. For example, the methods as they relate to identifying the status of the SNP rs71647871 in the CES1 genetic locus may be useful in assessing the ability of a patient to metabolize any one of a number of medications metabolized by CES 1. Exemplary drugs include, without limitation, clopidogrel, oseltamivir, methylphenidate, dexmethylphenidate, irinotecan, and the ACE inhibitors imidapril, enalapril, trandolapril, and ramipril.

[0035] The disclosure also provides a kit for detecting the SNP rs71647871 in the CES1 genetic locus, the kit comprising a set of at least two primer pairs suitable for practicing the methods described here, and one or more additional reagents and/or components, for example, a buffer solution, an enzyme such as a DNA polymerase, chain extension nucleotides such as deoxynucleotide triphosphates, a positive control sequence, and a negative control sequence.

[0036] By “threshold amount” is intended an amount, dose, concentration or level of a particular drug or compound of interest below which or above which the likelihood of one or more side effects in a particular population is altered. The threshold amount for different side effects may vary, may be comparable, or may be similar. Further it is recognized a particular drug or compound of interest may have a threshold amount that is the same or different from the threshold amount of another drug or compound of interest. Furthermore, a threshold amount may relate to one dose or bolus dose, an individual dose in a designated time period, multiple doses in a designated time period or a concentration in a sample from a patient.

[0037] Methylphenidate carries risk of side effects dizziness, drowsiness, changes in vision, heart problems, blood vessel problems, chest pain, trouble breathing, fainting, irregular heartbeat, tachycardia, increase in aggression, hostility, agitation, irritability, suicidal thoughts or behaviors, hallucinations, slow growth, weight loss, Reynaud’s phenomenon, tingling or pain in the extremities when exposed to cold, cold feeling in the extremities, skin color changes in fingers, priapism, xerostomia, insomnia, anxiety, tic disorder, nausea, headache, skin rash, hives, seizures, weight loss, runny nose, vomiting, hair loss. These potential negative side effects may be minimized by adjusting the dosing regimen. Often patients and their caregivers dislike side effects even including certain side effects that may appear minor. This dislike and frustration contributes to non- compliance with the dosing regimen such as reducing the dose, altering the dosing frequency or skipping the medication. Reducing administration of excess active compounds may reduce medical costs and the cost of prescription drugs to the patient or patient’s caregivers. On the other hand, under-dosing results in the administration of insufficient active ingredient to achieve the desired result, thus delaying treatment of the symptom or symptoms and increasing the amount of contact with the medical community the patient must have before seeing a positive result. Currently methylphenidate dosing regimens for patients with ADHD involve an initial dosing of about 2.5 mg, 5 mg, 10 mg, 15 mg, 18 mg, 20 mg or about 25 mg, followed by weekly gradual increases in dose to achieve a satisfactory result. It is recognized that methylphenidate dosing regimens for patients with different conditions may vary. For example, the dosing regimen for patients with narcolepsy may vary from the dosing regimen for patients with ADHD or ADD. A threshold amount of methylphenidate may range from about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 55 mg/day, to about 60 mg/day, about 65 mg/day, about 70 mg/day, about 72 mg/day, about 75 mg/day, about 78 mg/day, about 80 mg/day up to about 85 mg/day, about 90 mg/day, about 95 mg/day, about 100 mg/day, about 105 mg/day, about 110 mg/day, about 115 mg/day, about 120 mg/day, about 125 mg/day, about 130 mg/day, about 135 mg/day, about 140 mg/day, about 145 mg/day to about 150 mg/day.

[0038] Methylphenidate is prescribed to patients with a variety of indications, including but not limited to, ADHD and narcolepsy. By ADHD is intended to encompass attention-deficit/hyperactivity disorder, AD/HD, ADD, attention deficit disorder, ADHD predominantly combined type, ADHD predominantly inattentive type, and ADHD predominantly hyperactive-impulsive type.

[0039] Dexmethylphenidate is prescribed to patients with a variety of indications including, but not limited to ADHD and auditory processing disorders. Side effects of dexmethylphenidate include, but are not limited to, stomach upset, stomach pain, loss of appetite, dry mouth, headache, insomnia, restlessness, anxiety, feeling jittery, sore throat, increased blood pressure, increased heart rate, tachycardia, twitching and anorexia.

[0040] Currently dexmethylphenidate dosing regimens for patients with ADHD involve an initial dosing of about 2.5 mg, 5 mg, 10 mg, 15 mg, 18 mg, 20 mg or about 25 mg, followed by weekly gradual increases in dose to achieve a satisfactory result. It is recognized that dexmethylphenidate dosing regimens for patients with different conditions may vary. For example, the dosing regimen for patients with auditory processing disorder may vary from the dosing regimen for patients with ADHD. A threshold amount of dexmethylphenidate may range from about 5 mg/day, about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 55 mg/day, to about 60 mg/day, about 65 mg/day, about 70 mg/day, about 72 mg/day, to about 75 mg/day.

[0041] Oseltamivir is prescribed to patients with a variety of conditions including, but not limited to influenza, influenza A and influenza B. A threshold amount of oseltamivir may vary from about 20 mg/twice daily, 25 mg/twice daily, 30 mg/twice daily, 35 mg/ twice daily, 40 mg/ twice daily, 45 mg/ twice daily, about 50 mg/ twice daily, about 55 mg/twice daily, 60 mg/twice daily, 65 mg/twice daily, 70 mg/twice daily, 75 mg/twice daily, 80 mg/twice daily, 85 mg/twice daily, 90 mg/twice daily, 95 mg/twice daily, 100 mg/twice daily, 110 mg/twice daily, 120 mg/twice daily, 130 mg/twice daily, 140 mg/twice daily, to about 150 mg/ twice daily for 3, 4, 5, 6, 7, 8, 9, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days. Side effects of oseltamivir include but are not limited to toxic epidermal necrolysis, Stevens-Johnson Syndrome, erythema multiforme, self-injury, delirium, vomiting, diarrhea, and otitis media,

[0042] Trandolapril is prescribed to patients with hypertension, hypertensive disorders or who are at risk for hypertension. A threshold amount of trandolopril may vary from about 0.5 mg daily, 1 mg daily, 2 mg daily, 3 mg daily, 4 mg daily, 5 mg daily, 6 mg daily, 7 mg daily, 8 mg daily, 9 mg daily, 10 mg daily, 11 mg daily, 12 mg daily, 13 mg daily, 14 mg daily, 15 mg daily, 16 mg daily, 17 mg daily, 18 mg daily, 19 mg daily, to about 20 mg daily. Side effects of trandolapril may include, but are not limited to, intestinal angioedema, abdominal pain, nausea, vomiting, hypotension, oliguria, progressive azotemia, hyperkalemia, persistent cough, angioedema, syncope, neutropenia, headache, fatigue, and dizziness.

[0043] By efficacy is intended the effectiveness in treating a disease or disorder and improving a disease or disorder related symptom. Improvements in an influenza related symptom may include, but are not limited to alleviation of cough, alleviation of coryza, resolution of fever, and a return to normal health and activity. By reduced efficacy is intended a decreased effectiveness in treating a disease or disorder and limited improvement of a disease or disorder related symptom. Improvement, amelioration and bettering of a disease or disorder related symptom are encompassed.

[0044] ADHD and ADHD related symptoms may include, but are not limited to, problems with sustained attention, concentration and task persistence; overactivity, impulsivity, anxiety, inattentiveness, impatience, distractibility, forgetfulness, fidgeting, rapid movement, climbing, excessive talking, interruptive and disruptive. Influenza and influenza related symptoms may include, but are not limited to, flu and flu-related symptoms, chills, chilly sensation, fever, aches, pains, headache, photophobia, photoaversion, sore throat, dry cough, coryza, runny nose, nausea, vomiting, reduced sense of smell, fatigue and pneumonia. Hypertension and hypertension related symptoms may include, but are not limited to, hypertension, headache, fatigue, nausea, vomiting, shortness of breath, restlessness, blurred vision, drowsiness, confusion, seizures, sleepiness, abnormal heart rhythm, heart failure, excessive perspiration, tremor and paleness.

EXAMPLES

[0045] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It will be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0046] The following describes a method to accurately genotype rs71647871 status and its genomic context within the CES1 genetic locus. In particular, the methods described here are able to identify the status of rs71647871 and its location within either the allelic isoforms of CES1 (CES1 and CESlvar), or within the non-allelic isoforms (CES1P1 or CESlPlvar).

[0047] Patient genomic DNAs were extracted from buccal swabs using Mag- Bind® Blood and Tissue DNA HDQ 96 kit (Omega Bio-Tek, Norcross, GA, USA). A total of more than 60,000 U.S. de-identified patients were tested for CES1 143G/E genotyping. Cell line DNA was purchased from Coriell Institute for Medical Research (cat#: NA17237).

[0048] DNA sequences of the CES 1 isoforms were obtained from UCSC Genome Bioinformatics Center based on hgl9. CES1 and CESlvar share the same DNA sequence except in the 5’- untranslated region (5’UTR), exon 1 and intron 1. rs71647871 is located in exon 4. Two pairs of PCR primers were designed to amplify DNA fragments containing rs71647871. The first pair consisted of primers 12 and 13 which amplified a fragment of exon 4 from all four CES1 isoforms. The resulting PCR reaction product is designated as PCR A. The second pair consisted of primers 3 and 7 which amplified one fragment only from each of the allelic isoforms, CES1 and CESlvar, generating a 292 bp DNA fragment (designated as PCR B) comprising a unique identifying region.

[0049] Two different geno typing platforms were used to identify the status of rs71647871, either a mass spectrophotometry based platform (Mass ARRAY™, Agena Bioscience, Inc. of San Diego CA) or a PCR/primer extension platform (xTAG™ by Luminex). For the Mass Array™ platform, one extension primer was designed to specifically target rs71647871 in each of the PCR reactions, A and B. For the xTAG™ platform, two extension primers were designed, one each to target either the G or A nucleotide at position 428 of rs71647871. The claimed methods are not limited to either genotyping platform but may be practiced with any genotyping platform suitable for high throughput use.

MassARRAY iPLEX™ genotyping

[0050] SNP genotyping on a MassARRAY system (Agena Bioscience Inc., San Diego, CA) combines primer extension chemistry with highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). The genotyping method is based on the mass difference after the incorporation of one nucleotide into a target- specific extension primer. Target- specific DNA fragments were amplified by PCR reaction, and PCR products were dephosphorylated using shrimp alkaline phosphatase (SAP). A specific extension primer was added into the PCR products to effect a chain termination reaction. After removing excess salts by resin, the extended products were spotted onto a SpectroCHIP Array and detected by MALDI-TOF mass spectrometry using the manufacturer’s standard protocols. The incorporation of G or A onto an extension primer will have a mass difference of around 16 Daltons. Data were collected and analyzed with Type 4.0 software.

DNA fragments containing rs71647871 were amplified from each of the PCR A and B reactions using the extension primers. Extension products of the G (major/wild type) or A (minor/mutant) allele from each of the PCR A and B reactions were separated by absolute mass on MALDI-TOF. The results are shown as mass spectra (Fig. 1A) and clustered by allele ratio of log (Height). The peak height ratios of A/(A+G) from the two reactions indicate rs71647871 status as shown in Table 1 below. Table 1: rs71647871 status in PCR A and PCR B reactions based on peak height ratios

[0051] An rs71647871 status of homozygous wild type (G/G) is evidenced by a type G peak (Fig. 1A, Wild Type, G). Most normal heterozygotes (G/A) had both peaks in both of the PCR reactions, A and B. The A peak ratios were 25% in PCR A and 50% in PCR B (Heterozygote, G>A, normal het). This is concordant with the presumptive ratios (1 mutant out of 4 alleles in PCR A and out of 2 in PCR B). An rs71647871 status of homozygous mutant (A/A) is evidenced by a peak ratio of A/(A+G) about 50% for PCR A and 100% for PCR B (Homozygote, A) which is also concordant with presumptive ratios (2 mutants out of 4 alleles in PCR A and 2 mutants out of 2 alleles in PCR B).

[0052] During initial assay development and validation, we identified two minor groups of heterozygous alleles with unexpected ratios A/(A+G) of 25% and 36% and 25% and 100% respectively, as shown in Fig. IB and Table 2 below.

Table 2: Frequency of two interfering SNPs

[0053] The presence of an interfering SNP which resulted in failure to amplify one of the CES 1 alleles, resulting in a false report of homozygous instead of heterozygous rs71647871 during analysis was suspected. Additional experiments indeed showed that there was a G > A transition in rs71374094 on CES1P1 located in the 3’end of primer 3. This resulted in a PCR B reaction amplifying three isoforms, the two allelic isoforms CES1 and CESlvar and also a third isoform, CES IP 1, instead of only the two allelic isoforms, CES1 and CESlvar. This led to a call of 36% minor allele in PCR B instead of the expected 50% for heterozygous carriers. We also identified another interfering SNP in the form of a T > G transition in rsl50951789 in the 3’ end of primer 7. This led to a call of 100% minor allele in PCR B instead of the expected 50% for heterozygous carriers. Without being limited by mechanism, the interfering SNPs and resulting amplification issues may have contributed to the unsatisfactory results obtained from long-range PCR. xTAG™ genotyping

[0054] The xTAG™ platform detects single nucleotide polymorphisms by multiplex or single PCR followed with Allele Specific Primer Extension (ASPE) through Luminex’s Universal Tag sorting system on the Luminex 100/200 or FLEXMAP instruments. After PCR and Exonuclease I treatment to remove any leftover primers, biotin-labelled dCTP was incorporated during allele-specific primer extension (ASPE) which is specific to the G or A allele for each of the PCR A and B reactions. Specifically labelled target beads hybridize to and pull down the extended single strand DNA which is detected by streptavidin-phycoerythrin conjugated (SAPE) staining. The genotypes of each target are determined by sorting the beads by flow cytometry and measuring the fluorescent signal associated with each bead.

[0055] Median Florescence Intensity (MFI) for each allele is presented as numerical values in Table 3 A. MFIs are based on incorporation of biotin-labelled dCTP during the ASPE step, and a high MFI indicates presence of the specific A (MUT) or G (WT) allele. The absolute value and ratio of wild type and mutant were the criteria for data analysis. Genotype was determined by absolute MFI >1000 and the ratio (mut/mut+wt). For example, wild type CES1 ratios of mut/(mut+wt) were most commonly <10% and MFIs for WT were about 2000-3000 in both reactions. For reactions A and B heterozygous were 33-38% and 55-60%, respectively, and homozygous were 60% and 100%, respectively. For the heterozygous with rs 150951789, the ratio of mut/(mut+wt) was approximately 90%. The ratio of mut/(mut+wt) heterozygous with rs71374094, is -45%.

[0056] Table 3 A: Fuminex genotyping results from sample set 3. The MFI is presented for each sample ID, allele and reaction type. MUT: A allele MFI; WT: G allele MFI.

[0057] Table 3B: Ranges of A/(A+G) or MUT/(MUT+WT) ratios observed for tested samples. The ranges do not overlap and that allows for precise genotyping of CES1A1.

Prevalence of rs71647871 in population

[0058] SNPedia.com reports that the global minor allele frequency (GMAF) of rs71647871 is 0.01148. In the study from 63,830 patients, the minor allele frequency was 1.673%. According to the Hardy-Weinberg equation, p(G)=0.9833, q(A)=0.0186 leads to p+q=1.0019. Thus, the determined allele frequency fits well within the equation. Discussion

[0059] The rs71647871 SNP is located on Chromosome 16:55823658 (GRCh38) or 55857570(GRCh37) of exon 4 of the CES1 loci. There are currently no methods for the accurate detection of the homozygous minor allele of this SNP due to the complexity of the CES1 genetic locus. The method reported here addresses this need and can be used as a standalone assay or multiplexed with other assays to detect additional SNPs.

OTHER EMBODIMENTS

[0060] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0061] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0062] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:

1. A method for genotyping a single nucleotide polymorphism (SNP) in a biological sample from a subject, the SNP occurring within a genetic locus of a gene having multiple structural isoforms, the SNP consisting of a major allele and a minor allele, the method comprising: amplifying a first DNA fragment from the biological sample using a polymerase chain reaction (PCR) and a first pair of primers designed to amplify the first DNA fragment, the first DNA fragment consisting of portion of the gene which contains the SNP and which is the same in each of the multiple structural isoforms of the gene, to produce a first PCR reaction product, PCR A; amplifying a second DNA fragment from the biological sample using PCR and a second pair of primers designed to amplify the second DNA fragment, the second DNA fragment consisting of a portion of the gene unique to subset of the multiple structural isoforms, to produce a second PCR reaction product, PCR B; genotyping the SNP in each of the first and second PCR reaction products, PCR A and PCR B ; and comparing the relative amounts of the variant allele in each of PCR A and PCR B, thereby determining the status of the SNP within the subset of the multiple structural isoforms.

2. A method for genotyping the rs71647871 SNP in a biological sample from a subject and identifying its status within the four structural isoforms of the human CES 1 genetic locus, said locus consisting of two allelic isoforms, CES1 and CESlvar, and two non-allelic isoforms, CES IP and CESIPvar, the SNP consisting of a major allele (G) and a minor allele (A), the method comprising: amplifying a first DNA fragment from the biological sample using a polymerase chain reaction (PCR) and a first pair of primers designed to amplify the first DNA fragment, the first DNA fragment consisting of portion of the CES1 gene which contains the rs71647871 SNP and which is the same in each of the four structural isoforms of the gene, to produce a first PCR reaction product, PCR A; amplifying a second DNA fragment from the biological sample using PCR and a second pair of primers designed to amplify the second DNA fragment, the second DNA fragment consisting of a portion of the CES 1 gene unique to the allelic isoforms of the CES 1 gene, to produce a second PCR reaction product, PCR B ; genotyping the rs71647871 SNP in each of the first and second PCR reaction products, PCR A and PCR B; and comparing the relative amounts of the variant allele in each of PCR A and PCR B, thereby determining the status of the rs71647871 SNP within the allelic isoforms of the CES1 gene.

3. The method of claim 1 or 2, wherein the genotyping of the SNP is performed by a high throughput method.

4. The method of any one of claims 1-3, wherein the high throughput method comprises mass spectrophotometry or flow cytometry, or a combination of the two.

5. The method of any one of claims 1-4, wherein the method comprises a primer extension step.

6. The method of claim 4 or 5, wherein the mass spectrophotometry comprises matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF).

7. The method of claim 4 or 5, wherein the method comprises flow cytometry.

8. The method of claim 7, wherein the method comprises incorporating a labelled deoxyribonucleotide (dNTP) specific to the minor or major allele during a primer extension step and detecting the labelled dNTP in each of the extension PCR products A and B.

9. A kit of parts comprising a first pair of PCR primers and/or a second pair of PCR primers from any one of claims 1-8, and at least one additional reagent selected from the group consisting of a buffer, a detection reagent, a polymerase enzyme, a positive control sequence, and a negative control sequence.

10. The kit of claim 9, wherein the PCR primers are chemically modified.

11. The kit of claim 9 or 10, wherein the PCR primers are modified to contain a detectable label.

12. A method for treating a patient with methylphenidate, wherein the patient is suffering from ADHD, the method comprising the steps of:

(a) determining whether the patient is a CES1 poor metabolizer by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine if the patient has a CES1 poor metabolizer genotype; and if the patient has a CES 1 poor metabolizer genotype then internally administering methylphenidate to the patient a reduced amount of methylphenidate, and if the patient does not have a CES 1 poor metabolizer genotype, then internally administering methylphenidate to the patient in an amount that is greater than a threshold amount; wherein a risk of a side effect for a patient having a CES 1 poor metabolizer genotype is lower following the internal administration of said threshold amount than it would be if the methylphenidate were administered in an amount of greater than said threshold amount.

13. The method of claim 12, wherein said method of determining whether the patient has a CES 1 poor metabolizer genotype further comprises the steps of amplifying a first DNA fragment from said biological sample using a first pair of primers wherein said first DNA fragment comprises the portion of the CES1 gene which contains the rs71647871 SNP and consisting of or consisting essentially of a shared region to produce a first PCR reaction product; amplifying a second DNA fragment from the biological sample using a second pair of primers, wherein said second DNA fragment comprises a unique region of the CES 1 gene to produce a second PCR reaction product, wherein the unique region of the CES1 gene comprises a unique identifying sequence; genotyping the rs71647871 SNP in said first PCR reaction product; sequencing the second PCR reaction product; and comparing the relative amounts of the variant form of said first PCR reaction product to the relative amounts of each unique identifying sequence in said second PCR reaction product, thereby determining the source isoform of the rs71647871 SNP, and determining said patient has a CES 1 poor metabolizer genotype.

14. The method of claim 12, wherein the method of performing a genotyping assay is a bias neutral or bias corrected methods.

15. The method of claim 12, wherein said side effect is selected from the group comprising tachycardia, increased blood pressure, headache, nausea, vomiting, anxiety and seizures.

16. A method for treating a patient with dexmethylphenidate, wherein the patient is suffering from ADHD, the method comprising the steps of:

(a) determining whether the patient is a CES1 poor metabolizer by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine if the patient has a CES1 poor metabolizer genotype; and if the patient has a CES 1 poor metabolizer genotype then internally administering dexmethylphenidate to the patient a reduced amount of methylphenidate; and if the patient does not have a CES 1 poor metabolizer genotype, then internally administering dexmethylphenidate to the patient in an amount that is greater than a threshold amount, wherein a risk of a side effect for a patient having a CES 1 poor metabolizer phenotype is lower following the internal administration of said threshold amount than it would be if the dexmethylphenidate were administered in an amount of greater than said threshold amount.

17. The method of claim 16, wherein said method of determining whether the patient has a CES 1 poor metabolizer genotype further comprises the steps of amplifying a first DNA fragment from said biological sample using a first pair of primers wherein said first DNA fragment comprises the portion of the CES1 gene which contains the rs71647871 SNP and consisting of or consisting essentially of a shared region to produce a first PCR reaction product; amplifying a second DNA fragment from the biological sample using a second pair of primers, wherein said second DNA fragment comprises a unique region of the CES 1 gene to produce a second PCR reaction product, wherein the unique region of the CES1 gene comprises a unique identifying sequence; genotyping the rs71647871 SNP in said first PCR reaction product; sequencing the second PCR reaction product; and comparing the relative amounts of the variant form of said first PCR reaction product to the relative amounts of each unique identifying sequence in said second PCR reaction product, thereby determining the source isoform of the rs71647871 SNP, and determining if said patient has a CES1 poor metabolizer genotype.

18. The method of claim 16, wherein said side effect is selected from the group comprising tachycardia, anorexia, twitching and insomnia.

19. A method for treating a patient with oseltamivir, wherein the patient is suffering from influenza, the method comprising the steps of:

(a) determining whether the patient is a CES1 poor metabolizer by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine if the patient has a CES1 poor metabolizer genotype; and if the patient has a CES 1 poor metabolizer genotype then internally administering oseltamivir to the patient an increased amount of oseltamivir; and if the patient does not have a CES 1 poor metabolizer genotype, then internally administering oseltamivir to the patient in an amount up to a threshold amount, wherein a risk of a side effect for a patient that does not have CES 1 poor metabolizer phenotype genotype is lower following the internal administration of up to said threshold amount or less than it would be if the oseltamivir were administered in an amount of greater than said threshold amount.

20. The method of claim 19, wherein said method of determining whether the patient has a CES 1 poor metabolizer genotype further comprises the steps of amplifying a first DNA fragment from said biological sample using a first pair of primers wherein said first DNA fragment comprises the portion of the CES1 gene which contains the rs71647871 SNP and consisting of or consisting essentially of a shared region to produce a first PCR reaction product; amplifying a second DNA fragment from the biological sample using a second pair of primers, wherein said second DNA fragment comprises a unique region of the CES 1 gene to produce a second PCR reaction product, wherein the unique region of the CES1 gene comprises a unique identifying sequence; genotyping the rs71647871 SNP in said first PCR reaction product; sequencing the second PCR reaction product; and comparing the relative amounts of the variant form of said first PCR reaction product to the relative amounts of each unique identifying sequence in said second PCR reaction product, thereby determining the source isoform of the rs71647871 SNP, and determining said patient has a CES 1 poor metabolizer genotype.

21. The method of claim 19, wherein said side effect is selected from the group comprising vomiting and nausea.

22. A method for treating a patient with oseltamivir, wherein the patient is suffering from influenza, the method comprising the steps of:

(a) determining whether the patient is a CES1 poor metabolizer by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine if the patient has a CES1 poor metabolizer genotype; and if the patient has a CES 1 poor metabolizer genotype then internally administering oseltamivir to the patient an increased amount of oseltamivir; and if the patient does not have a CES 1 poor metabolizer genotype, then internally administering oseltamivir to the patient in an amount up to a threshold amount, wherein the efficacy of oseltamivir for a patient having a CES 1 poor metabolizer phenotype is lower following the internal administration of said threshold amount or less than it would be if the oseltamivir were administered in an amount greater than said threshold amount.

23. The method of claim 22, wherein said method of determining whether the patient has a CES 1 poor metabolizer genotype further comprises the steps of amplifying a first DNA fragment from said biological sample using a first pair of primers wherein said first DNA fragment comprises the portion of the CES1 gene which contains the rs71647871 SNP and consisting of or consisting essentially of a shared region to produce a first PCR reaction product; amplifying a second DNA fragment from the biological sample using a second pair of primers, wherein said second DNA fragment comprises a unique region of the CES 1 gene to produce a second PCR reaction product, wherein the unique region of the CES1 gene comprises a unique identifying sequence; genotyping the rs71647871 SNP in said first PCR reaction product; sequencing the second PCR reaction product; and comparing the relative amounts of the variant form of said first PCR reaction product to the relative amounts of each unique identifying sequence in said second PCR reaction product, thereby determining the source isoform of the rs71647871 SNP, and determining said patient has a CES 1 poor metabolizer genotype.

24. A method for treating a patient with trandolapril, wherein the patient is suffering from hypertension, the method comprising the steps of:

(a) determining whether the patient is a CES1 poor metabolizer by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine if the patient has a CES1 poor metabolizer genotype; and if the patient has a CES 1 poor metabolizer genotype then internally administering trandolapril to the patient an increased amount of trandolapril; and if the patient does not have a CES 1 poor metabolizer genotype, then internally administering trandolapril to the patient in an amount up to a threshold amount, wherein a risk of a side effect for a patient that does not have CES 1 poor metabolizer phenotype genotype is lower following the internal administration of up to said threshold amount than it would be if the trandolapril were administered in an amount of greater than said threshold amount.

25. The method of claim 24, wherein said method of determining whether the patient has a CES 1 poor metabolizer genotype further comprises the steps of amplifying a first DNA fragment from said biological sample using a first pair of primers wherein said first DNA fragment comprises the portion of the CES1 gene which contains the rs71647871 SNP and consisting of or consisting essentially of a shared region to produce a first PCR reaction product; amplifying a second DNA fragment from the biological sample using a second pair of primers, wherein said second DNA fragment comprises a unique region of the CES 1 gene to produce a second PCR reaction product, wherein the unique region of the CES1 gene comprises a unique identifying sequence; genotyping the rs71647871 SNP in said first PCR reaction product; sequencing the second PCR reaction product; and comparing the relative amounts of the variant form of said first PCR reaction product to the relative amounts of each unique identifying sequence in said second PCR reaction product, thereby determining the source isoform of the rs71647871 SNP, and determining said patient has a CES 1 poor metabolizer genotype.