Timal S 1 , Morava E 1 , Hoischen A 1 , Veltman J 1 , Wevers RA 1 , Lefeber DJ 1 1 Radboud Nijmegen Medical Centre, Nijmegen, Netherlands Identification of disease genes in patients with a Congenital Disorder of Glycosylation type I commonly follows a series of biochemical assays, several of those with limited availability. After extensive biochemical and genetic research, still 10% of CDG-I families in our cohort remain unsolved. Next-generation-sequencing techniques offer new alternatives to identify disease genes, thus far mostly applied to several families with identical clinical features. We hypothesized that the functional knowledge of a glycosylation abnormality in CDG-I patients would greatly facilitate gene identification in single cases. Whole-exome sequencing was applied on 6 single cases with CDG-I. After removal of known public and in-house polymorphisms,~600 private variants remained. A number of~60 possible CDG-I gene candidates was selected, based on their location in ER glycosylation, dolichol synthesis or cytoplasmic monosaccharide conversions. In 4 patients, the genetic cause of disease was identified: one as DPAGT1-CDG (a single case reported so far) and 2 novel gene defects. Mutation analysis and biochemical assays confirmed the pathogenicity of the identified variants. In the 2 remaining patients, no variants were found in the 60 candidate genes. This approach shows the huge potential to apply whole-exome-sequencing in metabolic disease, where functional knowledge of the disease is available. In a group of patients with dilated cardiomyopathy, a diagnosis of Congenital Disorders of Glycosylation (CDG) type I was found. After homozygosity mapping in the consanguineous families, pathogenic mutations were identified in DK1 in all individuals, while enzyme activity was deficient in fibroblasts. DK1 encodes the dolichol kinase responsible for formation of dolicholphosphate. In comparison with the severe multisystem presentation in CDG, the clinical presentation in our cohort was dominated by dilated cardiomyopathy, a rare feature in CDG. Expression analysis of DOLK in different tissues showed highest expression in fetal and adult brain, thereby not explaining the tissue specific phenotype. The availability of patient heart biopsy material allowed us to study other dolichol-phosphate dependent glycosylation pathways in affected tissue. N-glycosylation was affected as shown by western blotting of CD63. In addition, reduced O-mannosylation of alphadystroglycan was found with functional loss of its laminin-binding capacity. Loss of functional dystroglycan is known in a subgroup of the congenital muscular dystrophies, commonly presenting with dilated cardiomyopathy. Conclusions: Dolichol kinase deficiency results in decreased availability of dolichol-P-mannose thereby leading to abnormal O-mannosylation of alphadystroglycan. We thus describe a combined deficiency of protein N-glycosylation and O-mannosylation, presenting as late-onset dilated cardiomyopathy. Congenital disorders of glycosylation (CDG) are genetic diseases with an extremely broad spectrum of clinical presentations due to defective glycosylation of glycoproteins and glycolipids. Some 45 CDG types have been reported since the first clinical description in 1980. Protein glycosylation disorders are defects in protein N-and/or O-glycosylation. Dolichol phosphate is the carrier of the N-glycans during their assermbly first at the outside and subsequently at the inside of the endoplasmic reticulum (ER) membrane, and hence is a key molecule in protein glycosylation. Recently, defects have been identified in the last three steps of the dolichol phosphate biosynthesis: dolicholkinase deficiency (DK1-CDG), steroid 5alpha-reductase type 3 deficiency (SRD5A3-CDG), and dehydrodolichyl diphosphate synthase deficiency (DHDDS-CDG). We report on a patient with SRD5A3-CDG carrying a novel (homozygous) mutation. The diagnostic features of this novel inborn error of glycosylation are psychomotor retardation, nystagmus, visual impairment due to variable eye malformations, cerebellar abnormalities/ataxia, and often ichthyosiform skin lesions.