Genotyping assay to predict CYP3A5 phenotype

A method for determining the cytochrome P450 3A5 (CYP3A5) genotype and phenotype of an individual comprising:

• (a) isolating nucleic acid from the individual;
• (b) amplifying a region of the cytochrome P450 3A5 (CYP3A5) gene sequence selected from the group of:
• (i) intron 3 comprising the position corresponding to nucleotide 23 of SEQ ID NO:73;
• (ii) exon 7 comprising the position corresponding to nucleotide 29 of SEQ ID NO:74;
• and
• (iii) intron 3 comprising the position corresponding to nucleotide of SEQ ID NO:73 and exon 7 comprising the position corresponding to nucleotide 29 of SEQ ID NO:74;
• and
• (c) sequencing the amplified region of step (b), thereby determining the cytochrome P450 3A5 (CYP3A5) genotype and phenotype of the individual, wherein the cytochrome P450 3A5 phenotype is as follows:
• wherein the presence of an A at the position corresponding to nucleotide 23 of SEQ ID NO:73 on at least one CYP3A5 allele of said subject is indicative of a relatively high level of expression of CYP3A5 as compared to the presence of a G at that position;
• or the presence of a G at the position corresponding to nucleotide 23 of SEQ ID NO:73 on each CYP3A5 allele of said subject is indicative of a relatively low level of expression of CYP3A5 as compared to the presence of an A at that position;
• or
• wherein the presence of a G at the position corresponding to nucleotide 29 of SEQ ID NO:74 on at least one CYP3A5 allele of said subject is indicative of a relatively high level of expression of CYP3A5 as compared to the presence of an A at that position;
• or the presence of an A at the position corresponding to nucleotide 29 of SEQ ID NO:74 on each CYP3A5 allele of said subject is indicative of a relatively low level of expression of CYP3A5 as compared to the presence of a G at that position;
• or
• wherein the presence of an A at the position corresponding to nucleotide 23 of SEQ ID NO:73 and a G at the position corresponding to nucleotide 29 of SEQ ID NO:74 on at least one CYP3A5 allele of said subject is indicative of a relatively high level of expression of CYP3A5 as compared to the presence of a G at the position corresponding to nucleotide 23 of SEQ ID NO:73 and an A at the position corresponding to nucleotide 29 of SEQ ID NO:74 on at least one CYP3A5 allele of said subject;
• or the presence of either a G at the position corresponding to nucleotide 23 of SEQ ID NO:73 or an A at the position corresponding to nucleotide 29 of SEQ ID NO:74 on each CYP3A5 allele of said subject is indicative of a relatively low level of expression of CYP3A5 as compared to the presence of either an A at the position corresponding to nucleotide 23 of SEQ ID NO:73 or a G at the position corresponding to nucleotide 29 of SEQ ID NO:74 on each CYP3A5 allele of said subject.

A method of determining cytochrome P450 3A5 (CYP3A5) genotype of a subject which comprises

• (a) isolating nucleic acid from said subject;
• (b) amplifying a cytochrome P450 3A5 (CYP3A5) PCR fragment from said nucleic acid using a set of primers, wherein said set of primers contains primer X and primer Y;
• wherein
• i) primer X has the sequence of SEQ ID NO:
• 30, or a fragment thereof which is at least ten nucleotides long;
• and primer Y has the sequence of SEQ ID NO:
• 16, or a fragment thereof which is at least ten nucleotides long;
• or
• ii) primer X has the sequence of SEQ ID NO:
• 31, or a fragment thereof which is at least ten nucleotides long, and primer Y has the sequence of SEQ ID NO:
• 32, or a fragment thereof which is at least ten nucleotides long;
• and the amplified cytochrome P450 3A5 (CYP3A5) PCR fragment is in between primers X and Y;
• and
• (c) sequencing the amplified fragment obtained in step (b), thereby determining the cytochrome P450 3A5 (CYP3A5) exon 7 genotype of said subject.

A method for determining cytochrome P450 3A5 (CYP3A5) genotype of a subject which comprises:

• (a) isolating nucleic acid from said subject;
• (b) making a first and a second PCR primer wherein
• (i) the first PCR primer is complementary to exon 7 and introduces a base change in the PCR product produced by amplification with the first and second primer adjacent to or near the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7, such that a restriction site is generated in the presence of a particular nucleotide at the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7;
• and
• (ii) the second PCR primer is complementary to a region 3′ to the exon 7 nucleotide in the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7;
• (c) amplifying the sequence in between the first and the second primers;
• thereby obtaining an amplified fragment;
• and
• (d) treating the amplified fragment obtained in step (c) with a restriction enzyme in its corresponding restriction buffer to detect presence or absence of a point mutation at the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7, thereby determining the cytochrome P450 3A5 (CYP3A5) genotype of said subject.

A method for determining cytochrome P450 3A5 (CYP3A5) exon 7 genotype of a subject which comprises:

• (a) isolating nucleic acid from said subject;
• (b) amplifying a cytochrome P450 3A5 (CYP3A5) PCR fragment from said nucleic acid using a first set of primers, wherein said first set of primers contains primer X and primer Y;
• wherein
• (i) the X primer is complementary to a region 5′ to the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7;
• and
• (ii) the Y primer is complementary to a region 3′ to the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7;
• and the amplified cytochrome P450 3A5 (CYP3A5) PCR fragment is in between primers X and Y, thereby obtaining a first round amplified fragment;
• (c) amplifying the first round amplified fragment of step (b) using a second set of primers, wherein said second set of primers contains primer Z and primer W, wherein
• (i) primer Z is complementary to exon 7 and introduces a base change in the PCR product produced by amplification with the second set of primers adjacent to or near the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7, such that a restriction site is generated in the presence of a particular mutation at the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7;
• and
• (ii) primer W is complementary to a region 3′ to exon 7;
• and the amplified sequence is in between primers Z and W;
• and
• (d) treating the amplified fragment obtained in step (c) with a restriction enzyme in its corresponding restriction buffer to detect presence or absence of a point mutation at the position corresponding to nucleotide 29 of SEQ ID NO:74 in exon 7, thereby determining the cytochrome P450 3A5 (CYP3A5) genotype of said subject.