Nature Genetics
30, 233 - 237 (2002)
Published online: 14 January 2002; | doi:10.1038/ng826
Identification of a variant associated with adult-type hypolactasiaNabil Sabri Enattah1, Timo Sahi2, Erkki Savilahti3, Joseph D. Terwilliger4, Leena Peltonen1, 5
& Irma Järvelä1, 61 Department of Molecular Medicine, National Public Health Institute, Haartmaninkatu 8, PO Box 104, FIN-00251 Helsinki; and Department of Medical Genetics, University of Helsinki, Finland. 2 Department of Public Health, University of Helsinki. 3 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland. 4 Department of Psychiatry and Columbia Genome Center, Columbia University, New York, New York, USA; and Division of Medical Genetics, New York State Psychiatric Institute, New York, New York, USA. 5 Department of Human Genetics, UCLA School of Medicine, Los Angeles, California, USA. 6 HUCH-Laboratory Diagnostics, Laboratory of Molecular Genetics, Haartmanninkatu 2, PO Box 340, FIN-00290 Helsinki, Finland.
Correspondence should be addressed to lpeltonen@mednet.ucla.eduAdult-type hypolactasia, also known as lactase non-persistence (lactose intolerance), is a common autosomal recessive condition resulting from the physiological decline in activity of the lactase-phlorizin hydrolase (LPH) in intestinal cells after weaning. LPH hydrolyzes lactose into glucose and galactose. Sequence analyses of the coding and promoter regions of LCT, the gene encoding LPH, has revealed no DNA variations correlating with lactase non-persistence1,
2. An associated haplotype spanning LCT, as well as a distinct difference in the transcript levels of 'non-persistence' and 'persistence' alleles in heterozygotes, suggest that a cis-acting element contributes to the lactase non-persistence phenotype3,
4. Using linkage disequilibrium (LD) and haplotype analysis of nine extended Finnish families, we restricted the locus to a 47-kb interval on 2q21. Sequence analysis of the complete region and subsequent association analyses revealed that a DNA variant, C/T-13910, roughly 14 kb upstream from the LCT locus, completely associates with biochemically verified lactase non-persistence in Finnish families and a sample set of 236 individuals from four different populations. A second variant, G/A-22018, 8 kb telomeric to C/T-13910, is also associated with the trait in 229 of 236 cases. Prevalence of the C/T-13910 variant in 1,047 DNA samples is consistent with the reported prevalence of adult-type hypolactasia in four different populations. That the variant (C/T-13910) occurs in distantly related populations indicates that it is very old.
Lactase non-persistence limits the use of fresh milk among adults. The age of onset of lactase non-persistence ranges from 1−2 years among the Thai population to 10−20 years among Finns5,
6. In some populations, LPH activity persists throughout life in the majority of adults, a condition known as adult lactase persistence7. To identify the underlying DNA variation, we analyzed seven polymorphic microsatellite markers flanking LCT on 2q21 in nine extended Finnish families with hypolactasia (Fig. 1). We found significant evidence of linkage with markers D2S314, D2S442, D2S2196 and D2S1334; we obtained the highest lod score (7.67) at  =0 (= recombination fraction) with marker D2S2196 (Table 1). Recombination events defined the centromeric and telomeric boundary for the lactase persistence/non-persistence locus at markers D2S114 and D2S2385, respectively (Fig. 1 and Table 1).
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| | Table 1. Linkage and linkage disequilibrium in families with adult lactase non-persistence (the fine mapping markers are D2S3010−D2S3018) | | |  |
 Full Table |
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We assembled the draft sequence of the four BAC clones NH0034L23, NH0218L22, NH0318L13 and 329I10 (Fig. 2) into one uninterrupted 222.5-kb contig (Fig. 2). We used nine polymorphic markers in this region for the fine-mapping (Table 1). Six markers spanning a 200-kb region showed highly significant evidence of LD (P<10-4), whereas markers 3' of LCT showed no evidence of LD with lactase persistence alleles (Table 1). We constructed marker haplotypes over the critical interval for all alleles that were not identical by descent in the nine Finnish families, a total of 54 non-persistence alleles and 33 persistence alleles. One major haplotype was present in 20 persistence alleles (60%) versus 3 (5%) of the non-persistence alleles, whereas a wide diversity of haplotypes was seen in non-persistence alleles. The remaining 13 persistence alleles (40%) were detected in the ancestral haplotype, based on ancient recombinations (Fig. 3). The conserved haplotype restricted the critical region for lactase persistence to a 47-kb interval between markers D2S3013 and D2S3014. Consistent with our data, one major persistence haplotype has been reported in other Caucasian populations, such as British and French4,
8. Earlier efforts to monitor the common haplotype, however, focused solely on LCT and sequence in its immediate vicinity4.
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We analyzed the 47-kb region in DNA of seven family members, four with lactase persistence and three with non-persistence. The region contains only one known gene, MCM6 (ref. 9), that covers 36 kb of the critical 47 kb (Fig. 2). Although we did not find any variations in the coding region of MCM6, we identified a total of 52 non-coding variants: 43 SNPs and 9 deletion/insertion polymorphisms (Table 2). In the Finnish families, only two of the variants (C/T-13910, G/A-22018), 8 kb apart, showed complete cosegregation with the non-persistence trait (Tables 2 and 3). All family members with non-persistence were homozygous with respect to both C-13910 and G-22018 (Table 3). The C/T-13910 variant is 13,910 bp 5' of the initiation codon of LCT, in intron 13 of MCM6. The G/A-22018 variant is 22,018 bp upstream of the first ATG codon of LCT, in intron 9 of MCM6 (Fig. 2).
| | Table 2. Variations identified within Finnish families with a 47-kb adult lactase non-persistence locus | | | |
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| | Table 3. Distribution of genotypes in individuals with biochemically verified lactase persistence or non-persistence | | | |
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We analyzed these variants in Finnish DNA samples isolated from a total of 196 intestinal biopsy specimens with biochemically determined disaccharidase (LPH) activity. All 59 samples showing primary lactase deficiency were homozygous with respect to the C allele of C/T-13910, 6 were heterozygous with respect to the G/A-22018 variant and the remaining 53 were homozygous with respect to the G allele. Of the 137 cases showing lactase persistence, none were homozygous with respect to alleles C and G, at C/T-13910 and G/A-22018, respectively; 74 were homozygous for alleles T and A, with 63 being heterozygons at both positions. All 40 non-Finnish cases with verified disaccharidase deficiency (23 from South Korea, 9 from Italy and 8 from Germany) were homozygous (CC) with respect to C/T-13910. One Italian sample was heterozygous (GA) for G/A-22018, whereas the remaining 39 cases were homozygous (GG) at this position (Table 3).
To determine the prevalence of the lactase non-persistence−associated variant, we used the solid-phase minisequencing method10 to screen DNA samples of 938 anonymous Finnish blood donors originating either from the Western, early settlement or Eastern, late settlement regions of Finland. The overall prevalence of the lactase non-persistence genotype CC-13910 (170 cases) was 18.1%, with a lower prevalence in the sample from the western region (16.8%) than in the eastern region (18.9%; P=0.02, 1 df; Web Table A). These values are consistent with the results of an epidemiological study5.
The prevalence of lactase non-persistence in different populations varies greatly, from less than 5% in northern Europe to almost 100% in southeast Asia7. We determined the distribution of the genotype CC-13910 in 17 French people, 92 white North Americans and 96 African Americans. We observed the genotype CC-13910 in 41.2% of the French, in 7.6% of the white North Americans and in 79.2% of African Americans, in agreement with epidemiological data (Web Table A)11,
12.
The variant C/T-13910, completely associating with the non-persistence trait in both Finnish and non-Finnish samples, represents the most likely major variant underlying adult lactase non-persistence. The second variant, G/A-22018, is also fully associated with lactase persistence in the Finnish families, whereas in the case−control study sample, seven individuals with biochemically verified lactase non-persistence did not carry the associated genotype for this variant. However, we cannot exclude the possibility that both variants in the 8-kb haplotype would participate in the regulation of LPH activity.
Two hypolactasia-associated DNA variants reside at a considerable distance from LCT, and we can only speculate on their significance in the regulation of LPH activity. The C/T-13910 variant affects a binding site of transcription factor AP-2. The C allele, associated with lactase non-persistence, is in the consensus binding motif, whereas the T variant disrupts this motif. It is possible that the AP-2 element has a long-range cis-transcriptional effect on developmental stage−specific regulation of LCT. To establish the functional significance of this variant, further studies must control for transcriptional effects of all the other sequence variants (including the G/A-22018) in the relatively long 14-kb DNA segment 5' of LCT, and probably include the use of specialized cell lines, similar to the epithelial cells of the intestine.
We previously mapped13 the locus for congenital lactase deficiency (CLD) to an interval of approximately 2 cM 5' of LCT that spans the variants we describe here. We found no association between these variants and CLD in 19 Finnish families (data not shown).
A shared haplotype, the extent of LD in Finnish alleles and the presence of the same DNA variants in non-persistence alleles in different, distantly related populations together suggest that the persistence variant is old, occurring long before the differentiation of these populations. This is consistent with the hypothesis that adult lactase persistence has become more prevalent since the introduction of dairy culture in around 10,000−8,000 BC14. A selection power of 3−5% would be sufficient to explain the present frequency of the lactase persistence allele in northern Europe15, assuming it arose around the advent of dairy culture.
The unreliability of the indirect diagnostic methods of adult-type hypolactasia, such as the lactose tolerance test16, and the tedious measurement of LPH activity in jejunal biopsy sample specimens highlights the usefulness of a reliable DNA test. Large-scale epidemiological samples from different populations ascertained for biochemically verified hypolactasia should now be analyzed for the two DNA variants identified here. The outcome of these studies will determine the applicability of these variants as a diagnostic test for adult lactase non-persistence.
Methods
DNA samples analyzed.
The nine extended Finnish pedigrees have been described17. We tested 145 of 194 family members for lactase persistence, using lactose-tolerance tests with ethanol (LTTE)18. Gluten enteropathy was excluded in all lactose non-persistence cases by measurement of the serum IgA anti-tissue transglutaminase18. After obtaining informed consent, we extracted DNA from blood samples of all participating family members in accordance with standard protocols19. The case−control study sample represented 196 individuals with biochemically verified disaccharidase activities from jejunal biopsy specimens measured20 at the Helsinki University Hospital, all of them aged more than 20 y. In addition, DNA samples were available from nine Italian individuals (samples provided by M. Rossi, University of Naples), aged 35−65 y, nine German individuals (provided by M. Lentze, University of Bonn) aged 25−62 y and 22 South Korean individuals (provided by J.K. Seo, Seoul National University), aged 6−18 y, all of which had confirmed lactase non-persistence based on infestinal biopsy samples. One of the individuals from Germany originated from South Korea. Diagnosis was based on the measurement of disaccharidase activities in all but the South Korean samples, for which the diagnosis was confirmed by clinical symptoms. We determined the frequency of the C/T-13910 variant in 938 anonymous Finnish blood donors from Eastern and Western Finland and in 109 parents of families from Utah and France whose samples are kept by the Centre d'Etude Polymorphisme Humaine. The study was approved by the Ethical Committees of the Helsinki University Hospital and the Finnish Red Cross Blood Transfusion Service.
Genotyping.
We analyzed microsatellite markers flanking LCT on 2q21 as described elsewhere14. Microsatellite markers within the contig constructed over LCT were identified from the published genomic sequence of the BACs (NH034L23, NH0318L13, NH0218L22 and RP11-329I1) using the Repeat Masker program. We synthesized primers flanking the repeats and used PCR conditions as described elsewhere14. The amplified fragments were separated on 6% polyacrylamide gel.
Linkage and allelic association analyses.
We calculated pairwise lod scores using the MLINK option of the LINKAGE program package21,
22. We assumed autosomal recessive inheritance for adult-type hypolactasia with complete penetrance, no sex difference in recombination fractions and a disease allele frequency of 0.4. Only individuals older than 20 y were included in the study, as the condition is manifested by that age in the Finnish population7,
18. The affection status for individuals not confirmed by LTTE was regarded as unknown. We estimated allele frequencies and heterozygosity for the markers from family material, using the Downfreq program, to use in the parametric linkage analysis23. In addition, we carried out pseudomarker linkage and linkage disequilibrium analyses, assuming an autosomal recessive mode of inheritance, in which the frequencies of both disease and marker alleles are treated as nuisance parameters24. We carried out a test of LD conditional on the detected linkage, treating the allele frequencies and the recombination fraction as nuisance parameters23,
24. P values from these analyses are shown in Table 1. We constructed haplotypes using the program GENEHUNTER25.
Detection of the DNA variants.
We sequenced, for the critical 47-kb region, genomic DNA of three individuals with lactase non-persistence and four individuals with lactase persistence, representing phase-known chromosomal haplotypes in Finnish families. Using the published draft genomic sequence, we assembled to one contig the BACs (NH0034L23, NH0218L22 NH0318L23, RP-329I10) that covered the critical region of adult-type hypolactasia, using Sequencher 4 software (Gene Codes). We designed oligonucleotide primers spanning the critical region between them (a list of oligonucleotide primers are available on request). We carried out PCR amplification in a 50- l volume with genomic DNA (100 ng), primers (20 ng each), dNTPs (200 M), 0.5 U of Taq polymerase (Dynazyme, Finnzymes) in a standard buffer. Most PCR were amplified using the following PCR cycle conditions: an initial round of denaturation at 94 °C for 3 min, then 35 cycle at 94 °C at 30 s, 55 °C for 30 s, 72 °C for 1.25 min and a final extension of 72 °C for 10 min. In cases where the size of the PCR products was more than 1 kb, we used the Dynazyme extend kit. Purified PCR products (15−40 ng) were cycle-sequenced using BigDye terminator chemistry (PE Biosystems). We analyzed data using ABI Sequencing Analysis 3.3 (PE Biosystems) and Sequencher 4.1 (Gene Codes).
Screening of the lactase persistence/non-persistence variants.
We amplified the DNA fragment spanning the C/T-13910 variant using one biotinylated and one unbiotinylated primer. For G/A-22018 we used one biotinylated and one unbiotinylated primer under the conditions described above. Primer sequences are available upon request. We captured 10 l of the PCR product in a streptavidin-coated microtitre well (Labsystem, Finland) and carried out the minisequencing as described26. The minisequencing primers are available upon request. Two parallel minisequencing reactions were carried out for each PCR product.
URL.
Repeat Masker program, http://www.repeatmasker.org/.
GenBank accession numbers.
Markers used in the fine-mapping, AF395607−AF395615; BACs: NH0218L22, AC012551; N0034L34, AC011893; NH0318L13, AC011999; RP11-329I10, AC016516.
Note: Supplementary information is available on the Nature Genetics web site (http://genetics.nature.com/).
Received 6 September 2001; Accepted 18 December 2001; Published online: 14 January 2002.
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Acknowledgments
We are grateful to the families who participated in this study. We thank M. Levander for assistance with minisequencing. This work was supported by The Academy of Finland, The Paediatric Research Foundation (Ulla Hjelt Fond), The Sigrid Jusélius Foundation and Helsinki University Hospital Research Funding and The National Institutes of Health (grant to J.D.T.).
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3' direction. The positions of ten polymorphic microsatellite markers used for fine mapping of the locus are given. The backslashes in the horizontal line denote gaps in the contig sequence. The position of marker D2S2169 was confirmed by bridging the gap with PAC 106O20 isolated from the PAC library. The organization of MCM6 is shown below, including the position of the lactase persistence−associated variants in introns 9 and 13, located 13.9 kb and 22 kb 5' of the first ATG of LCT.
