Gene identification in the congenital disorders of
glycosylation type I by whole-exome sequencing
Sharita Timal1,2, Alexander Hoischen3, Ludwig Lehle5, Maciej Adamowicz6,
Karin Huijben2, Jolanta Sykut-Cegielska7, Justyna Paprocka8, Ewa Jamroz8,
Francjan J. van Spronsen9, Christian Ko ¨rner10, Christian Gilissen3, Richard J. Rodenburg2,
Ilse Eidhof2, Lambert Van den Heuvel2, Christian Thiel10, Ron A. Wevers2, Eva Morava4,
Joris Veltman3and Dirk J. Lefeber1,2,∗
1Department of Neurology,2Department of Laboratory Medicine,3Department of Human Genetics and4Department of
Pediatrics, Institute for Genetic and Metabolic Disease, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands5Department of Cell Biology and Plant Biochemistry, University of Regensburg, Regensburg, Germany,
6Department of Biochemistry and Experimental Medicine, The Children’s Memorial Health Institute, Warsaw, Poland,
7Department of Metabolic Diseases, Children’s Memorial Health Institute, Warsaw, Poland,8Child Neurology
Department, Medical University of Silesia, Katowice, Poland,9Beatrix Children’s Hospital, University Medical Center of
Groningen, University of Groningen, Groningen, The Netherlands and10Center for Child and Adolescent Medicine and
Center for Metabolic Diseases Heidelberg, Department Kinderheilkunde I, Heidelberg, Germany
Received February 10, 2012; Revised March 26, 2012; Accepted March 28, 2012
Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic dis-
eases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms
and the large number of potential genes involved. Until now, gene identification included the sequential
application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homo-
zygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic
strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome
sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene iden-
tification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous fam-
ilies. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the
rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked
CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together
the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleo-
tide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity
of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been
identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in
the CDG-I in newly diagnosed singleton families.
Genetic defects in the glycosylation of proteins and/or lipids
result in a large and rapidly growing group of neurometabolic
diseases, collectively called the congenital disorders of
glycosylation (CDG). Of all glycosylation pathways, protein
N-glycosylation is best understood (Fig. 1). The first part of
the pathway, i.e. dolichol-glycan assembly and glycan transfer
∗To whom correspondence should be addressed at: Department of Neurology, Laboratory of Genetic, Endocrine and Metabolic Disease, Institute for
Genetic and Metabolic Disease, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands.
Tel: +31 243614428; Fax: +31 243618900; Email: email@example.com
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to nascent proteins in the endoplasmic reticulum (ER), involves
a multistep process and is identical for each N-glycosylated
protein. Genetic defects in this process cause CDG type I
(CDG-I). Patients often present with ahighlyvariablemultisys-
tem phenotype, including neurological symptoms such as psy-
chomotor retardation, muscle hypotonia, seizures, liver and
kidney symptoms, endocrine and coagulation abnormalities
and variable dysmorphic features (1,2). Due to their variability,
clinical features are not discriminative for the many possible
defects in the glycosylation route (3). Only in a few cases,
genetic defects lead to specific clinical features, such as muscu-
lar dystrophy in DPM3-CDG (4), that can be used as a diagnos-
tic criterion. The large number of possible genes involved in
cing approach for the identification of causative gene defects.
The current strategy for the identification of new disease-
causing genes in CDG-I patients has heavily relied on yeast
alg mutants and the functional knowledge of their involvement
in the N-glycosylation pathway (5). A combined genetic-
biochemical approach using homozygosity mapping in
consanguineous families was successfully applied to identify
novel genes in the N-glycosylation process (6,7). Altogether,
these approaches have resulted in 17 genetically distinct
CDG-I subtypes currently known (CDG-Ia to Iq, in the
former nomenclature) that can be classified into four different
functional groups (Fig. 1): dolichol-phosphate synthesis and
recycling (A), synthesis and transport of nucleotide sugars
(B), dolichol-linked oligosaccharide biosynthesis (C) and the
oligosaccharyltransferase complex (D).
Whole-exome sequencing (WES) offers new opportunities
to quickly identify disease genes in Mendelian disorders
(8–11). Disease genes were successfully identified by priori-
tizing potential variants based on the data of homozygosity
mapping and linkage analysis, or combining results from mul-
tiple patients with an identical clinical phenotype (12,13) or
from patient–parent trios (14). The identification of disease
genes in single cases remains difficult, although there has
been some success for autosomal recessive disorders (15,16).
Especially in single cases, the definitive annotation of a muta-
tion as pathogenic is extremely challenging and requires the
identification of independent cases with similar mutations or
a functional confirmation. In this respect, glycosylation disor-
ders have the advantage that functional assays can readily be
chosen on the basis of the identified candidate gene.
unsolved CDG-I patients from our cohort of 117 cases with
different clinical phenotypes and from non-consanguineous
parents. On the basis of the functional knowledge of the glyco-
sylation process, a CDG-I gene list (Supplementary Material,
CDG-I patient, which led to the identification of four of
six defects. This shows the potential to apply WES for gene
discovery in CDG-I patients.
Patients and clinical presentation
In our cohort of 117 CDG-I patients, six non-related patients
without known consanguinity remained unsolved. The patients
represented a diverse clinical spectrum as briefly summarized
in Table 1. Patient 3 was an adopted child of the Colombian
Figure 1. Glycan assembly pathway. Four main groups of proteins involved in the glycan assembly in the N-glycosylation pathway: (A) dolichol synthesis;
(B) synthesis and transport of nucleotide sugars; (C) dolichol-linked oligosaccharide biosynthesis and (D) oligosaccharyltransferase complex.
4152Human Molecular Genetics, 2012, Vol. 21, No. 19
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