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A novel proximal -13914G>A base replacement in the vicinity of the common-13910T/C lactase gene variation results in an atypical LightCycler melting curve in testing with the mutareal lactase test.

To the Editor:

The common -13910T/C exchange located 13910-bp upstream of the lactase gene (LCT) is associated with the persistence /nonpersistence trait of adult-type hypolactasia, which leads to symptoms of lactose malabsorption, including bloating, flatulence, diarrhea, and abdominal pain. The typing of this variant is used as a genetic test for lactase (non)-persistence. We used the MutaReal Lactase real-time PCR test (Immundiagnostik) with the Roche LightCycler capillary system to analyze genomic DNA and observed in 1 sample an aberrant melting curve profile attributable to a novel base replacement, -13914G>A, in the vicinity of the common -13910T/C lactase gene variation.

Lactose intolerance results primarily from the physiological decline in activity of the LCT gene-encoded enzyme lactase-phorozin hydrolase, which breaks down milk sugar. Use of linkage disequilibrium and haplotype analysis of affected Finnish families revealed a defined DNA transition (-13910T/C) proximal to the LCT gene in intron 13 of the adjacent minichromosome maintenance type 6 gene (MCM6), which is associated with verified lactase nonpersistence and reduced bone mineral density predisposing for bone fractures (1, 2). Persons homozygous for C are at higher risk for lactose intolerance but may not show the typical symptoms. Nevertheless, genetic testing for the base-pair replacement is being offered as a genetic test for lactase persistence. The MutaREAL Lactase test is a real-time PCR for detection of this single-nucleotide polymorphism (SNP). A 222-bp PCR product is generated from the regulatory region of the lactase gene and analyzed by melting curve analysis. Typically, the genotype T/T has a [T.sub.m] of 52[degrees]C and the C/C variant of 61[degrees]C with a variance of [+ or -] 1[degrees]C, and the heterozygous genotype has a biphasic melting curve with [T.sub.m]s at both temperatures.

According to the national guidelines approved by the German Medical Association, we routinely used the MutaREAL Lactase test to genotype patients who consulted the internal medicine or outpatient departments with symptoms of adult-type hypolactasia. In 121 samples, 26 patients (22%) were genotyped to T/T and 52 (43%) to C/T, and 42 (35%) were homozygote carriers of the C-allele associated with the lactose intolerance. This frequency, higher than the ~15%-20% in the general population in Germany (3), supports the linkage of this SNP with lactose intolerance, as established by specific experiments (4).

Genotyping of a 37-year-old male patient revealed an atypical, previously unreported melting curve (Fig. 1) displaying 2 peaks, with [T.sub.m]s at 52[degrees]C and 57[degrees]C. Although the [T.sub.m] at 52[degrees]C was typical for the T variant, the T. at 57[degrees]C represented neither the C nor the T variant. We performed standard PCR to amplify and sequence the 383-bp fragment spanning the corresponding region (initial denaturation at 94[degrees]C for 3 min, then 35 cycles at 94[degrees]C for 1 min, 57[degrees]C for 1 min, 72[degrees]C for 1 min, and final extension at 72[degrees]C for 10 min) with primers LacUl (5'-GAA TGC TCA TAC GAC CAT GG-3') and LacR1 (5'-CTG CTT TGG TTG AAG CGA AG-3'). This PCR generated the predicted fragment in all 3 controls (T/T, C/T, and C/C) and the patient sample (see Fig. 1 in the Data Supplement that accompanies the online version of this Letter at Amplicons were purified, subjected to cycle sequencing with fluorescent dye terminators using primer LacU2 (5'-GGT AAG CAT TTG AGT GTA GTT G-3'), and analyzed on an ABI PRISM 310 automatic sequencer (PE Applied Biosystems). The analysis revealed that the patient was heterozygous for the -13910T/C variation and carried a previously unreported transition from G to A at position -13914 in one allele (see Fig. 2 in the online Data Supplement). We then cloned and sequenced respective allele fragments and found that the C at -13910 was linked to an A at position -13914 and the Tat-13910 to a G at-13914 (see Fig. 3 in the online Data Supplement).


DNA fragments with the C and T variants in the -13910T/C region may enhance LCT promoter activity (5). The substitution at -13914 is not known to affect lactase activity, but the sequence 5'-GAT AAT GTA GC/T CCC TGG CCT C-3' surrounding the -13910 variant contains a binding site for an unidentified specific, differentially interacting, transactivation factor that can enhance lactase transcription in human intestinal cells (5). In addition, the -13910T/C variant is located in the MCM6 gene, which encodes a eukaryotic replication factor implicated and required for both initiation and elongation of chromosomal DNA replication. Further investigation of these variants may shed light on their clinical significance.

We report a genetic variation identified with fluorescent hybridization probes and melting analysis conducted on the LightCycler system. The increasing numbers of these reports suggest the need for an online database for aberrant melting curves and their underlying variations.

DOI: 10.1373/clinchem.2006.077529


(1.) Enattah NS, Sahi T, Savilahti E, Terwilliger JD, Peltonen L, Jarvela I. Identification of a variant associated with adult-type hypolactasia. Nat Genet 2002;30:233-7.

(2.) Obermayer-Pietsch BM, Bonelli CM, Walter DE, Kuhn RJ, Fahrleitner-Pammer A, Berghold A, et al. Genetic predisposition for adult lactose intolerance and relation to diet, bone density, and bone fractures. J Bone Miner Res 2004;19: 42-7.

(3.) Buning C, Ockenga J, Kruger S, Jurga J, Baier P, Dignass A, et al. The C/C(-13910) and G/G(22018) genotypes for adult-type hypolactasia are not associated with inflammatory bowel disease. Scand J Gastroenterol 2003;38:538-42.

(4.) Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Jarvela I. Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia. Gut 2003;52:647-52.

(5.) Olds LC, Sibley E. Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element. Hum Mol Genet 2003;12:2331-40.

Carmen G. Tag Marie-Claire Schifflers Monika Mohnen Axel M. Gressner Ralf Weiskirchen *

Institute of Clinical Chemistry and Pathobiochemistry RWTH-University Hospital Aachen, Germany

* Address correspondence to this author at: Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, D-52074 Aachen, Germany. Fax 49-241-80-82512; e-mail
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Title Annotation:Letters
Author:Tag, Carmen G.; Schifflers, Marie-Claire; Mohnen, Monika; Gressner, Axel M.; Weiskirchen, Ralf
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jan 1, 2007
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