Here we report four new mutations in the LCT
gene underlying congenital lactase deficiency (CLD). The mutations were identified in compound heterozygous form in three patients (Italian and Finnish) with characteristic features of CLD. There are three kinds of evidence that these mutations lead to CLD 1) the substitutions change the amino acid sequence of a protein in a conserved region of the LPH protein, 2) substitutions were predicted to be damaging by several prediction methods and 3) healthy control subjects lack these mutations. We also found mutation G1363S in a homozygous form in two siblings of Turkish origin. This mutation has earlier been reported in one Finnish CLD patient in eastern (near the Russian border) Finland suggesting that the mutation may have been brought to Finland from the east [10
]. This is the first time that CLD cases and the underlying mutations have been identified outside Finland.
Mutations E1612X and R1587H are located in the conserved region IV of the mature LCT
that encodes lactase activity (Figure ) [20
]. Mutation E1612X leads to a truncated protein. In substitution R1587H both amino acids are polar, basic amino acids. However, arginine always has a positive charge and a role in the maintenance of protein overall charge balance. Therefore the substitution to histidine (which charge depends on environment) may disturb the overall charge. Substitution S688P and deletion V565fsX567 are located in the region II of the pro-LPH that has been shown to have a role as an intramolecular chaperone in the folding of the LPH-protein (Figure ) [23
]. Deletion V565fsX567 leads to truncated protein after a few amino acids. A change from a non-aromatic polar serine to cyclic and non-polar proline causes a remarkable change in the chemical and physical properties of the protein.
Schematic presentation of the LCT gene and locations of the mutations. Cleavage sites are R734/L735 and R867/A868.
All four novel mutations were examined by several prediction programs [17
]. Although the Blosum62 matrix does not take into account the conservation of the protein region, the results showed that there is low probability of both substitutions (serine to proline and arginine to histidine) of occurring in the functional protein. In PolyPhen the prediction was based on PSIC score (logarithmic ratios of likelihood of a studied amino acid occurring at a particular site to the likelihood of same amino acid occurring at any site) [18
]. Since multiple alignment profile scores provide a major contribution to the prediction, it remains reasonably reliable even without the missing 3-D information of LPH. [18
]. Using PolyPhen both substitutions (R1587H and S688P) were considered to be pathogenic. The results obtained with SIFT or PANTHER PSEC were in line with the results above.
Taken together, a total of nine mutations are known to underlie CLD to date. The mutations are quite evenly distributed covering both the pro region and the mature LPH (Figure ). Location of mutations does not affect the severity of the phenotype. In contrast, all mutations lead to a severe phenotype. The phenotype of CLD is in striking contrast with the phenotype of adult-type hypolactasia, associated with the normal down-regulation of lactase activity with mild or absent symptoms. It is noteworthy that in these two conditions, the values of lactase activity are partially overlapping in spite of their having different underlying molecular mechanism [2
]. However symptoms are much more severe in CLD.
After the identification of the molecular background of CLD, genetic testing has been offered in Finland. Interestingly, nine novel CLD cases were identified in 2006 and two in 2007. All patients were diagnosed at the first weeks of age based on severe watery diarrhoea and loss of weight. Nine of them were homozygous for the major mutation Y1390X further confirming the original results that only one major mutation is enriched in the Finnish population [10
]. Two patients were heterozygous for the founder mutation. In both of them (case 2 and 3) new mutations were reported here. Earlier, CLD has been estimated to be very rare with a frequency of 1:60 000 newborns in Finland [5
]. The figures presented here suggest that CLD may be more common than previously estimated. CLD should be suspected in neonates with severe diarrhoea which starts after introduction of milk feeding. A high concentration of lactose is present in liquid faeces and may easily be identified. The diarrhoea is cured within hours by substituting milk with a lactose-free formula. Later, CLD may be diagnosed by either genetic testing or by studying disaccharidase concentrations in the duodenal mucosa.