Genetic test for detecting congenital adrenal hyperplasia

A method for amplifying a CYP21A2 gene and/or a CYP21A2 gene chimera from a sample, comprising:

• a) amplifying a product from a sample comprising human genomic DNA by PCR using:
• i. a forward primer that is complementary to a sequence that is duplicated in a bimodular human RCCX locus, wherein the bimodular human RCCX locus comprises, in order, an RP1 gene, a C4A gene, a CYP21A1P pseudogene, a TNXA gene, an RP2 gene, a C4B gene, a CYP21A2 gene and a TNXB gene, and:
• the first occurrence of the duplicated sequence is in the C4A gene and/or the RP1 gene;
• and
• the second occurrence of the duplicated sequence is in the C4B gene and/or the RP2 gene;
• and
• ii. a reverse primer that is complementary to a sequence that occurs only once in the bimodular human RCCX locus at a position in the TNXB gene, wherein:
• the product of (a) contains the sequence of the CYP21A2 gene but not the sequence of the CYP21A1P pseudogene if the human genomic DNA comprises a CYP21A2 gene that has not been rearranged;
• and,
• the product of (a) contains the sequence of the CYP21A1P/CYP21A2 gene chimera if the human genomic DNA comprises an RCCX locus that has been rearranged to produce a CYP21A1P/CYP21A2 gene chimera;
• and
• b) amplifying a plurality of segments of the product of (a) by PCR using multiple primer pairs, wherein the primer pairs hybridize to sequences that are in both the CYP21A2 gene and the CYP21A1P pseudogene, wherein at least some of the amplified segments are overlapping, wherein the amplifying is done in:
• i) a first multiplex PCR reaction that comprises some but not all of the primer pairs;
• and
• ii) a second multiplex PCR reaction that comprises the remainder of the primer pairs;
• wherein:
• the segments amplified by the first multiplex PCR reaction are not overlapping;
• the segments amplified by the second multiplex PCR reaction are not overlapping;
• and
• at least some of the segments amplified by the first multiplex PCR reaction overlap with the segments amplified by the second PCR reaction.