Mutations in the translated region of the lactase gene (LCT) underlie congenital lactase deficiency

Abstract

Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. We initially assigned the CLD locus by linkage and linkage disequilibrium on 2q21 in 19 Finnish families. Here we report the molecular background of CLD via characterization of five distinct mutations in the coding region of the lactase (LCT) gene. Twenty-seven patients out of 32 (84%) were homozygous for a nonsense mutation, c.4170T-->A (Y1390X), designated "Fin(major)." Four rare mutations--two that result in a predicted frameshift and early truncation at S1666fsX1722 and S218fsX224 and two point mutations that result in substitutions Q268H and G1363S of the 1,927-aa polypeptide--confirmed the lactase mutations as causative for CLD. These findings facilitate genetic testing in clinical practice and enable genetic counseling for this severe disease. Further, our data demonstrate that, in contrast to common adult-type hypolactasia (lactose intolerance) caused by a variant of the regulatory element, the severe infancy form represents the outcome of mutations affecting the structure of the protein inactivating the enzyme.

Figure 1

CLD haplotypes ordered according to each identified mutation. Patients 14, 16, 17, 27, and 32 are compound heterozygous for Y1390X and their respective rare mutations. Microsatellite mutation of D2S314 in one patient increased the total number of CLD chromosomes to 49. Microsatellite markers were searched for and ordered on the basis of genetic maps of the Marshfield Clinic Research Foundation, as well as the physical map of the UCSC Genome Browser (December 2001 assembly). The Baylor College of Medicine (BCM) Search Launcher repeat-masker algorithm was used to identify novel microsatellites from clones between D2S314 and D2S2385 (Smith et al. 1996). Finally, 15 microsatellites and C/T₋₁₃₉₁₀, a SNP associated with adult-type hypolactasia, were analyzed, covering 5.88 cM of the CLD region. DNA was extracted by a standard protocol (Vandenplas et al. 1984) from the peripheral blood samples. PCR was performed and the genotypes were assigned as described elsewhere (Ylisaukko-oja et al. 2004). Haplotypes were constructed using the GENEHUNTER 2.1 program (Kruglyak et al. 1996). The CLD founder haplotype was generated with the assumption that there were a minimum number of recombinations.

Figure 2

Transcript map of the critical DNA region for CLD. The physical map was constructed using public (International Human Genome Sequencing Consortium) Human Genome Project annotations. Evidence for transcripts and for conserved amino acids was searched with BLAST against different databases, including the dbEST and Refseq protein databases. Complete sequence annotations were performed by the NIX program (UK Human Genome Mapping Project Resource Centre) and the UCSC Genome Browser. LCT, minichromosome maintenance deficient 6 (MCM6 [MIM 601806]), aspartyl-tRNA synthetase (DARS [MIM 603084]), chemokine receptor type 4 (CXCR4 [MIM 162643]), and KIAA1679 are the known genes in the region (horizontal arrows). The mouse ortholog (BC063250) of human KIAA1679 suggests that ESTs BX954188 and BX954180 may be related to transcripts of KIAA1679. Microsatellite markers are indicated by vertical arrows. The centromere is on the left.

Figure 3

CLD mutations in the LCT gene. A–E, DNA sequence chromatograms of five identified CLD mutations. One mutation, Y1390X, (A) is a homozygote, whereas the rest of the mutations are heterozygotes. The first row shows affected sequences and the second row wild-type sequences. In addition, c.655G→A, a SNP (rs3754689) leading to V219I, is shown in panel E (Boll et al. 1991). PCR was performed using the genomic DNA-amplifying promoter region, exons, flanking intron sequences, and 3′-UTRs of the LCT gene (primers are available on request). Sequencing was performed in both directions, and the sequenced products were electrophoresed on an ABI 3730 DNA analyzer (Applied Biosystems) in accordance with the manufacturer’s instructions and were analyzed using ABI Sequencing Analysis 3.3 (Applied Biosystems) and Sequencher 4.1 (Gene Godes).

Figure 4

Structure of lactase and the location of the identified CLD mutations. The genomic size of the LCT gene is ∼55 kb and is composed of 17 exons (Boll et al. 1991). The size of messenger RNA (mRNA) is 6,274 bases, and the primary translation product (prolactase) is 1,927 amino acids. The prolactase contains a cleavable signal sequence from Met₁ to Gly₁₉ that guides the polypeptide to the endoplasmic reticulum (von Heijne ; Mantei et al. 1988). The region from Ser₂₀ to Thr₁₈₈₂ consists of four homologous domains (I–IV). The prolactase is processed by two proteolytic cleavages. The first, an intracellular cleavage, occurs between Arg₇₃₄ and Leu₇₃₅. The second cleavage in the intestinal lumen between Arg₈₆₈ and Ala₈₆₉ generates lactaseβ_final, the mature enzyme (Jacob et al. ; Wüthrich et al. 1996). The figure is modified from Naim (2001).

Figure 5

Carrier frequency of Y1390X in four subpopulations in the early and later settlement regions of Finland. The carrier frequencies of the CLD mutations were determined from 556 anonymous blood donors obtained from the Finnish Red Cross Blood Transfusion Service.