Simultaneous genotyping of the three lactose tolerance-linked polymorphisms LCT-13907C>G, LCT-13910C>T and LCT -13915T>G with Pyrosequencing™ technology

Background: We required a new genotyping method for the diagnosis of adult hypolactasia, which would allow the simultaneous genotyping of three known polymorphic loci linked to lactose tolerance (LCT -13907C>G, LCT-13910C>T and LCT-13915T>G) in a single PCR/pyrosequencing test run. Method: We utilized Pyrosequencing™ technology, a DNA-sequence-by-synthesis technique. Results: The developed Pyrosequencing™ method allowed genotyping of the three loci LCT-13907C>G, LCT -13910C>T and LCT -13915T>G in a single PCR/pyrosequencing test run. A separate Pyrosequencing™ assay was developed for genotyping of the LCT -14010G>C mutation. The methods were evaluated in 116 clinical samples from patients of non-European descent, sent to the laboratory for diagnosis of adult hypolactasia. The African mutations LCT -13907C>G and LCT-13915T>G were found in subjects originating not only from Somalia, Ethiopia and Eritrea but also in Arabs and Iranians. Several compound heterozygotes LCT-13907CG/-13915TG were found among Ethiopian, Eritrean and Somalian subjects. No subject with the LCT-14010G>C mutation was found among the studied subjects. Advantages compared to the other genotyping methods are less staff hands-on time than, e.g., restriction fragment length polymorphism (RFLP) analyses, avoiding radioactivity as in the originally described isotope-minise-quencing and in addition, Pyrosequencing™ is a direct DNA sequencing technique which gives unambiguous genotyping results as well as some redundant sequence information beyond the single nucleotide polymorphism (SNP) position, which serves as a valuable internal control obtained for each sample. Conclusions: Pyrosequencing™ is a robust genotyping modality suitable for clinical genotyping of patients not only of European, but also of African or Middle Eastern descent, who may harbor any combination of the three LCTmutations, LCT-13907C>G, LCT-13910C>T, LCT-13915T>G.