Transcriptional downregulation of the lactase (LCT) gene during childhood

Adult-type hypolactasia, characterised by bloating, gas formation, and diarrhoea after ingestion of lactose containing food, affects half of the world’s population.¹ The molecular background of lactase non-persistence/persistence trait has been shown to associate with a single nucleotide polymorphism (SNP) C/T₋₁₃₉₁₀ residing 13910 base pairs upstream from the 5′ end of the lactase (LCT) gene in an intron of the minichromosome maintenance 6 (MCM6) gene.^2–4 We have demonstrated a trimodal distribution of lactase activity in the intestinal mucosa in adults, with low lactase activity (4–9 U/g protein) in those with the C/C₋₁₃₉₁₀ genotype.³ The C₋₁₃₉₁₀ and T₋₁₃₉₁₀ allele show differential regulation of lactase promoter activity and binding capacity for the nuclear proteins in electromobility shift assay.^5,6 Our recent analysis in a paediatric population demonstrated that the main time period for lactase downregulation in Finns and in Somalians is from five to 10 years of age.⁴

To further assess the role of the C₋₁₃₉₁₀ allele in downregulation of lactase activity during development, we isolated lactase RNA from intestinal biopsy samples with verified disaccharidase activities.⁷ The study group comprised 15 subjects aged 10 months to 23 years, 12 with the C/T₋₁₃₉₁₀ genotype and three with the C/C₋₁₃₉₁₀ genotype. All subjects were heterozygous for the G/A₊₅₉₃ polymorphism residing in exon 1 of the LCT gene. Relative expression of lactase mRNA transcribed from the C₋₁₃₉₁₀ and T₋₁₃₉₁₀ allele was assessed by quantitative minisequencing using the G/A₊₅₉₃ polymorphism on the LCT gene as a marker. The methods used are described in detail by Kuokkanen and colleagues.³ The study was approved by the ethics committee at the Helsinki University Central Hospital. All families gave their informed consent.

Subjects with the C/T₋₁₃₉₁₀ genotype (age range 10 months to 17 years) had high lactase activity, ranging from 21 to 113 U/g protein (mean activity 47 U/g protein; sample not available n = 2) except for one child presenting with low lactase activity (6 U/g protein). In this case the indication for endoscopy was exclusion of gastro-oesophageal reflux. Due to the very low lactase activity and the C/T₋₁₃₉₁₀ genotype, mutations underlying congenital lactase deficiency (CLD)⁸ were screened for this patient and he was shown to be a carrier of a CLD mutation (unpublished data). Of the three subjects with the C/C₋₁₃₉₁₀ genotype, the oldest subject aged 23 years had low lactase activity (6 U/g protein) as expected; the five year old subject had high lactase activity (24 U/g protein). Lactase mRNA was transcribed in a 1:1 ratio from the C₋₁₃₉₁₀ and the T₋₁₃₉₁₀ allele in children younger than five years of age. In children over six years of age, relative lactase mRNA expression from the C₋₁₃₉₁₀ allele was reduced to 18% and 16% compared with that from the T₋₁₃₉₁₀ allele (fig 1 ▶, table 1 ▶).

Figure 1.

 Relation of age to relative expression of lactase LCT mRNA from the C₋₁₃₉₁₀ compared with that from the T₋₁₃₉₁₀ allele. Actual relative expression of the C allele in the biopsy sample was obtained by relating the results of the minisequencing to an 11 point standard curve (y = 0.0135× − 0.9714 up to 82%; y  =  1×10⁻⁵e^0.1129x for 82–100%) obtained based on information of the relative amounts of G and A in a G/A₊₅₉₃ heterozygous genomic DNA sample (for details of methods, see Kuokkanen et al³).

Table 1.

 Lactase activity, L/S ratio, and allelic ratio of the study subjects

Age (y)	C/T−13910 genotype	Lactase activity (U/g protein)	L/S ratio	Allele ratio (%) C/T−13910*
0.8	CT	85	1.11	48/52
1.1	CT	113	1.02	52/48
4.0	CT	31	0.49	48/52
4.3	CT	53	0.48	42/58
4.7	CT	40	0.62	40/60
4.9	CT	–	–	42/59
5.1	CT	6	0.08	48/52†
6.7	CT	22	0.28	18/82
7.6	CT	–	–	16/84
11.1	CT	29	0.54	13/87
14.9	CT	21	0.40	17/83
17.0	CT	29	0.62	24/76
1.1	CC	–	–	52/48
5.0	CC	24	0.28	51/49
22.8	CC	6	0.08	49/51

*Defined by assessing cSNP G/A₊₅₉₃ in exon 1 of the lactase LCT gene.

†Carrier of a CLD mutation (unpublished data).

Our results show an increasing imbalance in relative mRNA expression levels of the C₋₁₃₉₁₀ and T₋₁₃₉₁₀ alleles in children aged >5 years. These results support the earlier findings on transcriptional regulation of the lactase gene^9,10 and the finding in our own laboratory that the persistent T₋₁₃₉₁₀ allele was shown to represent a mean of 92% of expressed lactase mRNA in C/T₋₁₃₉₁₀ heterozygous adults.³ The decline in lactase mRNA expression transcribed from the C₋₁₃₉₁₀ allele in the intestinal mucosa occurs in parallel with the time period of the decline in lactase enzyme activity, indicating a causative role for the intronic region containing the C₋₁₃₉₁₀ allele. Characterisation of the transcriptional regulators at the C/T₋₁₃₉₁₀ enhancer element and the exact mechanism underlying C₋₁₃₉₁₀ allele specific downregulation of lactase activity awaits elucidation.

Acknowledgements

We are grateful to the children and their families for their participation. Ms Sari Näsman and Mervi Mannonen at the Day Surgery Unit, Hospital for Children and Adolescents, are acknowledged for coordinating and managing the sample collection.

Funding was provided by the Sigrid Jusélius Foundation, Helsinki, Finland, the Helsinki University Hospital Research Funding, Helsinki, Finland, the Finnish Cultural Foundation, the Maud Kuistila Foundation, and The Research Foundation of Orion Pharma, Espoo, Finland.