Autosomal recessive dilated cardiomyopathy due to DOLK mutations results from abnormal dystroglycan O-mannosylation

Abstract

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.

Figure 1. Pedigrees, haplotypes, and mutation analysis of families I through IV.

Upper panels: segregation analysis of the homozygous region at 9q33.1-qter. Black bars represent haplotypes that segregate with the disease. Lower panels: chromatograms of affected family members showing the c.1222C>G, c.912G>T, and c.3G>A mutations (upper profile) and of controls (lower profile). Mutated base pairs and corresponding amino acid residues are printed in bold.

Figure 2. Histology and immunohistochemistry of heart sections of patient II/5 stained with haematoxylin eosin (HE), alpha dystroglycan clone IIH6C4 (IIH6), beta dystroglycan (β-dys), beta-sarcoglycan (β-sar), and desmin.

PR: right ventricle of patient, PL: left ventricle of patient, C: control. Bar = 0.05 mm.

Figure 3. CDG biochemistry.

A. N-glycosylation abnormalities in affected patients as observed by isoelectrofocusing of serum transferrin. 0, 2 and 4 indicate asialo-, disialo-, and tetrasialotransferrin isoforms. C: control, PMM2: PMM2-CDG. B. Dolichol kinase activity in patient (n = 5) and control (n = 7) fibroblasts. Microsomal fractions were incubated with γ³²P-CTP and dolichol-19, and dolichol-³²P production was measured.

Figure 4. Functional analysis of DOLK mutant alleles in the temperature sensitive yeast sec59 mutant.

A. Serial dilutions of sec59 mutant, transformed with plasmids, harboring either wild-type DOLK, mutant alleles, or empty vector, were plated on YPD media and incubated at 25, 32, or 37°C. B. Glycosylation pattern of CPY in sec59 mutant transformed with wild-type and mutant DOLK alleles. Cells were cultivated at the permissive temperature of 25°C and shifted for 10 h to 32 or 37°C, followed by immunoblotting as described . The positions of mature CPY (mCPY) and underglycosylated forms lacking one to four N-glycans are indicated.

Figure 5. Expression of dolichol kinase by mRNA expression analysis in human fetal and adult tissues.

Relative expression levels are given as the fold change in comparison to the tissue with the lowest expression level.

Figure 6. Western blotting for analysis of O-mannosylation and N-glycosylation.

O-mannosylation (A) and N-glycosylation (B). A. Heart muscle homogenates were used with (left panel) or without (right panel) WGA-enrichment and stained for desmin, β-dystroglycan, β-sarcoglyan, glycosylated α-dystroglycan (IIH6) and used for functional analysis of laminin binding. PR: patient II/5 right ventricle; PL: patient II/5 left ventricle; HC: healthy control; PC: pathological cardiomyopathy control. B. Patient and control fibroblasts and heart tissue homogenates were used for western blotting of CD63. Lanes 1 and 4: control; lane 2: DPM1-CDG; lane 3: DOLK-CDG; lane 5: patient right ventricle; lane 6: patient left ventricle.

Figure 7. Involvement of dolichol kinase (DOLK) in N-glycosylation and O-mannosylation in the ER.

The enzymes involved in O-mannosylation of alpha-dystroglycan are indicated. Dystroglycan is proteolytically cleaved into a beta and alpha subunit. The mucin domain of α-DG contains the O-mannosyl glycans that are required for binding to the basal lamina via laminin. In addition, mucin O-glycans and phosphorylated O-mannosyl glycans (not shown) are present.