High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants

Abstract

Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT −13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase persistence can be associated with other variants nearby the −13910C>T variant, limiting the use of the −13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the −13915T>G variant among 78 patients genotyped as −13910C/C by a TaqMan assay. All samples originated from patients of probable Middle Eastern descent. In order to detect the −13910 and −13915 variants simultaneously, we developed a new high-resolution melting (HRM) analysis assay based on unlabeled probe genotyping and simultaneous amplicon scanning analysis. By using this assay we were able to distinguish the −13910 and −13915 genotypes clearly. Furthermore, we identified two rare variants, the −13907C>G and −13913T>C. With this method, based on an inexpensive unlabeled probe, it is possible to simultaneously detect the −13910C>T and −13915T>G variants in addition to rarer variants surrounding the −13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance.

Key Words::

• Lactase persistence/non-persistence
• genotyping
• lactose intolerance
• LCT variants
• molecular diagnostics
• mutation
• SNP
• high-resolution melting

Acknowledgements

The expert technical assistance of Kirsten Hald and Kirsten Kruse Olsen is appreciated. The critical reading of the manuscript by Anne Dorthe Møller is warmly appreciated.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.