A defect in dolichol phosphate biosynthesis causes a new inherited disorder with death in early infancy

Abstract

The following study describes the discovery of a new inherited metabolic disorder, dolichol kinase (DK1) deficiency. DK1 is responsible for the final step of the de novo biosynthesis of dolichol phosphate. Dolichol phosphate is involved in several glycosylation reactions, such as N-glycosylation, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and C- and O-mannosylation. We identified four patients who were homozygous for one of two mutations (c.295T-->A [99Cys-->Ser] or c.1322A-->C [441Tyr-->Ser]) in the corresponding hDK1 gene. The residual activity of mutant DK1 was 2%-4% when compared with control cells. The mutated alleles failed to complement the temperature-sensitive phenotype of DK1-deficient yeast cells, whereas the wild-type allele restored the normal growth phenotype. Affected patients present with a very severe clinical phenotype, with death in early infancy. Two of the patients died from dilative cardiomyopathy.

Figure 1.

A, Pedigree of the family with the c.295T→A mutation, showing the high degree of consanguinity. Affected patients are indicated by blackened squares, proven heterozygotes by half-blackened symbols. B, Clinical presentation of DK1 deficiency. The 6-mo-old girl (ASB) showed profound muscular hypotonia, inflammation and ichthyosis of the skin, nearly complete secondary loss of hair, and severe DCM.

Figure 2.

Transferrin IEF, which shows increased amounts of disialo- and asialotransferrin in the patients. Numbers designate the number of carbohydrate side chains.

Figure 3.

Incorporation of [³H]-glucosamine into LLOs. Fibroblasts were labeled for 30 min with 100 mCi [6-3H]glucosamine per ml labeling medium, and dolichol-linked oligosaccharides were extracted as described. Incorporation of [³H]-glucosamine was related to total protein. Controls were set to 100%.

Figure 4.

DK1 assay of patient fibroblasts. The reaction mixtures (A) consisted of 100 mM Tris-HCl buffer (pH 7.4), 30 mM CaCl₂, 20 mM UTP, 6 μM CTP (3,000 Ci/mmol–specific activity), and 5 μg of dolichol, previously suspended in Triton X-100 (0.1% final concentration). The reaction was started by the addition of up to 700 μg of crude cell extracts, to a final volume of 100 μl, and was stopped after 20 min by the addition of 800 μl of chloroform/methanol (2:1 v/v). After dolichol phosphate (Dol-P) was extracted, the samples were dried under nitrogen and were measured in a scintillation counter. The activity of the patients' crude extracts were severely reduced (residual activity [±SD]: NB 3.2%±1.8%; ASB 4%; AYB 3.9%±1.6%) compared with control cell lines. B, Assay samples, separated by TLC. Pig Dol-P was used as standard.

Figure 5.

Growth characteristics of transformed Sec59 yeast cells, showing 10-fold dilutions from top to bottom. Pictures were taken after 1 wk at the permissive temperature (30°C) (left) or at the restrictive temperature (37°C) (right). From left to right in both panels: cells transformed with human wild-type allele (wt), cells transformed with the c.1322A→C allele (for subjects ASB and AYB), cells transformed with the c.295T→A allele (for subjects NB and GH), and mock-transformed cells.