Background: Diagnosis of lactose intolerance is commonly based solely on the absence of the European lactose tolerance allele MCM6 -13910T, although other lactose tolerance mediating alleles exist, especially in defined African populations and in the Middle East. Therefore, a SimpleProbe assay was developed which can simultaneously detect -13910T as well as common African variants.
Methods: A SimpleProbe oligonucleotide was used to discriminate between three classes of MCM6 alleles: I: Intolerance (-13910C); II: Caucasian type tolerance (-13910T); III: African/Arabian type tolerance (-13907G, -13913C, -13915G). Genotyping standards for all these alleles were constructed through PCR amplification and cloning from genomic DNA. Assay performance was tested using real-time PCR and melting curve analyses with genotyping standards as templates. Finally, the genotyping accuracy was tested using two panels: 100 individuals, previously genotyped using Sanger-sequencing and 20 individuals, previously genotyped using TaqMan analyses.
Results: The assay showed a clear discrimination between gene variants conferring lactose tolerance and intolerance. Mean Tmvalues for the Caucasian alleles -13910C and -13910T were 55.3 °C and 60.8 ºC respectively, whereas Tm-values for all three African/Arabian variants (-13907G, -13913C and -13915G) were in the interval of 50-52 ºC. SimpleProbe genotyping of both panels showed 100 % concordance with the results of the reference methods.
Conclusions: The new SimpleProbe LightCycler assay provides a rapid, cost effective and reliable method to diagnose lactose intolerance and is especially suited for populations with African or Middle Eastern admixture.

Keyword:lactose tolerance, MCM6 gene, African variants, SimpleProbe LightCycler assay

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