TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

ABSTRACT

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.