Single tube multiplex PCR method for the detection of HBA1, HBA2 and HBA12 genes
One aspect of the invention is a method for amplifying alpha globin genes HBA1, HBA2 and HBA12 in a single PCR tube to determine an HBA genotype of a subject. This method employs five primers selected to accurate and sensitively identify the HBA1, HBA2, and HBA12, a gene found at a higher frequency in citizens of Saudi Arabia, by accurately annealing to nucleic acids in a biological sample and simultaneously amplifying sequences encoding the alpha globin genes. This invention includes a procedure and required reagents for the amplification of alpha globin genes in a single PCR tube.
1. A method for treating a subject at risk of a hemoglobin A (HBA) associated disease or trait by detecting Hemoglobin alpha 1 (HBA1) gene, Hemoglobin alpha 2 (HBA2) gene, and Hemoglobin alpha 12 (HBA12) genes in the subject, comprising:
obtaining a sample from the subject;
contacting the sample with at least one set of primers for each of HBA1, HBA2 and HBA12 under conditions suitable for the primer sets to amplify DNA in the sample by multiplex polymerase chain reaction (PCR), wherein the set of HBA1 primers are MA1F (SEQ ID NO: 3) and MA12R (SEQ ID NO: 4), the set of HBA2 primers are MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO: 2 or 6) and the set of HBA12 primers are MA12F (SEQ ID NO: 1) and MA2R (SEQ ID NO: 2 or 6),
detecting the presence of, or levels of, HBA1, HBA2 and HBA12 DNA amplified by each primer set, thereby detecting a genotype of the subject, and
treating a course of an HBA-associated disease or treating a trait using a detected genotype in combination with clinical data to inform a treatment decision.
2. The method of claim 1 , wherein said subject carries gene conversion HBA12.
3. The method of claim 1 , wherein said subject does not carry gene conversion HBA12.
4. The method of claim 1 , wherein the sample is blood, plasma or serum, or white blood cells, or cells obtained from a buccal swab.
5. The method of claim 1 , wherein the sample is cells obtained from a buccal swab.
6. The method of claim 1 , wherein the primers for HBA1, HBA2 and HBA12 are part of a single primer pool.
7. The method of claim 1 , further comprising identifying the subject as being at risk of sickle cell disease when the presence of amplified HBA1, HBA2 and HBA12 is detected.
8. The method of claim 1 , further comprising identifying the subject as having sickle cell trait or sickle cell disease when the presence of amplified HBA1, HBA2 and HBA12 is detected.
9. The method of claim 1 , further comprising identifying the subject as being at risk of sickle cell disease when the presence of amplified HBA1, HBA2 and HBA12 is detected and treating the subject for sickle cell disease with at least one of penicillin, hydroxyurea, a transfusion of red blood cells, or by hematopoietic stem cell transplantation.
10. The method of claim 1 , further comprising identifying the subject as being at risk of thalassemia when the presence of amplified HBA1, HBA2 and HBA12 is detected.
11. The method of claim 1 , further comprising identifying the subject as being at risk of thalassemia when the presence of amplified HBA1, HBA2 and HBA12 is detected and treating the subject for thalassemia with at least one of a blood transfusion, iron chelation therapy, or folic acid.
12. A method for genetic counselling comprising the method of claim 1 , wherein said treating a course of an HBA-associated disease or trait using the detected genotype in combination with clinical data comprises informing the subject of his or her HBA-associated gene associations.
13. The method of claim 12 , wherein said genetic counseling is premarital counseling.
14. The method of claim 12 , wherein said subject is identified as having an a12/a1, a12/a1 genotype.
15. The method of claim 12 , wherein said subject is identified as having an a12/a1, a12/-- genotype.
16. The method of claim 12 , wherein said subject is identified as having an a12/--, a12/-- genotype.
17. The method of claim 12 , wherein said subject is identified as having an a12/--, --/-- genotype or a --/--, --/-- genotype.
18. A method for differential diagnosis comprising the method of claim 1 , wherein said treating a course of an HBA-associated disease or trait using the detected genotype in combination with clinical data comprises informing a health care provider of the subject's HBA-associated gene associations with sickle cell disease or with thalassemia.
19. The method of claim 18 , wherein said subject carries gene conversion HBA12.
20. The method of claim 18 , wherein said subject does not carry gene conversion HBA12.