ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy that is accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of diseases associated with abnormal post-translational processing of a-dystroglycan that share a defect in laminin-binding glycan synthesis1. Although mutations in six genes have been identified as causes of WWS, only half of all individuals with the disease can currently be diagnosed on this basis2. A cell fusion complementation assay in fibroblasts from undiagnosed individuals with WWS was used to identify five new complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the ISPD gene (encoding isoprenoid synthase domain containing). The pathogenicity of the identified ISPD mutations was shown by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin-binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a new mechanism for WWS pathophysiology.

The hallmark of dystroglycanopathies - and the common pathogenic denominator in all WWS (MIM# 236670) patients - is loss of functional glycosylation of α-dystroglycan (α-DG)[3]. Lack of proper α-DG glycosylation reduces binding to extracellular matrix proteins since the ligand binding is mediated through the α-DG sugar moiety[3,4]. All six of the causative genes previously identified in WWS patients demonstrate autosomal recessive inheritance and code for known or putative glycosyltransferases; mutations therein result in abnormal α-DG glycosylation[5]. Nevertheless, approximately half of the WWS patient population has no mutations in these known genes[2], emphasizing the need for WWS gene discovery.

We established a complementation assay to enable us to identify additional genes that contribute to WWS pathology. This assay was developed based on a panel of fibroblasts from six genetically defined, but heterogeneous dystroglycanopathy cases (Supplementary Table 1), for which western blotting revealed a lack of functional glycosylation (defined as immunoreactivity to IIH6, a monoclonal antibody specific for requisite sugar moiety) and an inability to bind laminin (Fig. 1a). The results are consistent with previously published data obtained from various dystroglycanopathy patient tissues[3,6] and cells[7-9]. Notably, the degrees of α-DG hypoglycosylation varied by the gene mutated, and the molecular weight of α-DG produced in each case is hypothesized to reflect an abnormality at different steps in α-DG glycan biosynthesis (Fig. 1a). For each patient population the α-DG glycosylation defect was rescued by transferring a wild-type copy of the mutant gene. For example, in WWS fibroblasts with known POMT1 mutations, α-DG functional glycosylation was restored by adenovirus-mediated gene transfer of POMT1, but not by expression of the other known WWS genes (Fig. 1b). This complementation assay was adapted to On-Cell western blotting, and rescue of α-DG functional glycosylation was demonstrated for all known WWS genes (Fig. 1c). Previously, it was shown that forced overexpression of the putative glycosyltransferase LARGE can induce α-DG hyperglycosylation in control cells and bypass the α-DG glycosylation defect in dystroglycanopathy patient cells[8,10]. However, we now demonstrate that the ability of LARGE to hyperglycosylate α-DG is dependent on the availability of O-mannosyl phosphate acceptor sites and correlates with the severity of the clinical phenotype (Fig. 1d).

Fig. 1

α-DG glycosylation status in dystroglycanopathy patient dermal fibroblasts

WGA-enriched cell lysates from control and patient fibroblasts (samples with defects in six different genes, Supplementary Table 1) were subjected to biochemical analysis. (a) α-DG glycosylation status as assessed by western blotting using antibodies against the glycosylated form of α-DG (IIH6), core α-DG (G6317), and by laminin overlay assay. An antibody against β-DG (AP83) was used to assess loading. Apparent molecular masses are indicated. (b) Complementation assay. Immunoblot of POMT1 deficient -WWS cells infected with a panel of adenoviruses expressing WWS candidate genes; only adenovirus mediated gene transfer with a POMT1wild-type copy rescues the defect. (c) On-Cell western analysis of fibroblasts from control and genetically heterogeneous WWS patients demonstrates that the complementation approach, using adenovirus mediated gene transfer, can successfully be applied to all known WWS genes. Restoration of the glycosylation defect is indicated with a red circle. Bottom: sample from a non-diagnosed WWS patient; lack of rescue suggests a novel genetic defect. Note, LARGE overexpression rescues and bypasses the α-DG hypoglycosylation in less severe WWS-MEB cells with hypothesized residual activity (POMT2 and POMGnT1), but not in the most severe loss-of-function WWS patient cells. The On-Cell western blot was probed with antibodies against the glycosylated form of α-DG (IIH6) and for signal enhancement the cells were co-infected with dystroglycan expressing adenovirus (Ad5CMV-DAG1). (d) Quantitative On-Cell western blot analysis of LARGE-induced α-DG hyperglycosylation in control and POMT1 deficient cells from three patients with different clinical severity. The ability of LARGE to increase the affinity of the cell surface for the IIH6 antibody and bypass the glycosylation defect in POMT1 deficient patient cells correlates with the residual activity of the mutant gene product and the severity of the clinical manifestation. IIH6 On-Cell quantitative data were normalized with DRAQ5 cell DNA dye (n=3). Error bars represent s.d.

We next applied the On-Cell complementation assay to fibroblasts from a cohort of 63 dystroglycanopathy patients (Supplementary Fig. 1), identifying 11 WWS individuals from 10 unrelated families, who we postulated to have mutations in novel genes. Our first step toward defining the genetic basis for WWS in these patients was to establish complementation groups; to this end we adapted a cell fusion approach that is commonly used in yeast research[11] and has proven successful in mammalian cells[12]. Whereas fusion is achieved by mating in the case of yeast cells, polyethylene glycol (PEG)[13] treatment is used for mammalian cells. We hypothesized that fusion between co-cultured cells from patients harboring recessive mutations in the same gene would not rescue the α-DG glycosylation defect, whereas fusion between cells from patients with independent genetic defects would rescue successfully. Immunofluorescence analysis revealed IIH6-positive fused cells (as indicated by the presence of multiple nuclei) only when two cell lines from genetically different patients were co-cultured; complementation between POMT1 and FKTN fibroblasts is shown in Fig. 2a. Two-way fusions of WWS cells with mutations in each of the known genes rescued α-DG glycosylation (data not shown). Application of the PEG fusion approach to all 11 genetically unidentified WWS patients led to the identification of five separate complementation groups (Fig. 2b,c), suggesting that five novel WWS genes are represented in this small cohort of patients. While four complementation groups were represented by a single WWS patient, one group consisted of seven WWS individuals. If mutations in a single novel gene are responsible for disease in all seven patients, this complementation group should represent a relative common cause of WWS.

Fig. 2

Cell fusion experiments reveal novel genetic complementation groups

Cell fusion among co-cultured dermal fibroblasts induced with polyethylene glycol (PEG). (a) Immunofluorescence-based detection of restored α-DG functional glycosylation with glyco α-DG antibodies (IIH6) (scale bar 50μm). In contrast to cultures containing POMT1-WWS or FKTN-WWS patient cells only, co-cultures of these cells exhibited restored functional α-DG glycosylation in multinucleated cell fusions. Nuclei are stained with DAPI. (b) Fibroblasts from control and different WWS patients (five) with unknown genetic defects were co-cultured and subjected to cell-fusion complementation. Complementation was assessed as rescue of functional α-DG glycosylation, using glycosylation-specific α-DG antibodies (IIH6) by On-Cell western blotting. Rescue of the glycosylation defect is indicated by red circles. For signal enhancement the cells were co-infected with dystroglycan expressing adenovirus (Ad5CMV-DAG1). (c) Schematic representation summarizing our identification of five new complementation groups. Red arrows indicate successful fusion complementation. One group contains seven patients, whereas the remaining four groups currently only consist of a single patient.

All seven patients within this complementation group met the classic diagnostic criteria for WWS (Supplementary Table 2). Two of the cases, P5[6,14] and P6[15], were previously published and described as WWS. In the case of P1, brain MRI examinations performed at 3 days and 5 months of age, showed hydrocephalus, cobblestone lissencephaly of the cerebral cortex, severe brainstem hypoplasia with a kink at the isthmus, and severe hypoplasia of the cerebellum (Fig. 3a). This patient also displayed evidence of severe muscular dystrophy (Fig. 3b), bilateral microophthalmia with cataracts and arrested retinal development. Immunofluorescence and western blot analysis of a skeletal muscle biopsy from this patient showed that this new complementation group of WWS patients manifests the typical α-DG glycosylation defect in skeletal muscle with loss of both functional glycosylation and receptor function (Fig. 3b,c). Comparative analysis of the α-DG glycosylation status in fibroblasts from five different WWS cases in the same complementation group confirmed that all samples share a defect in α-DG processing, with complete loss of functional glycosylation and laminin binding (Supplementary Fig. 2). Moreover, the loss of post-translational modification and a subsequent shift to lower molecular weight was comparable in all samples, consistent with the hypothesis that they share a common genetic defect.

Fig. 3

Clinical presentation and α-DG glycosylation defect in ISPD-WWS patient P1

(a) Sagittal and axial T1 MRI brain images at 5 months of age showed severe ventriculomegaly, agyria and a significantly malformed (Z-shaped) hypoplastic brainstem, as well as severely hypoplastic cerebellar vermis. In addition, the axial image reveals subcortical heterotopia. (b) Histologic staining of frozen cross-sections from a skeletal muscle biopsy, showing severe dystrophic histopathology with muscle fiber necrosis and regeneration, as well as endomysial fibrosis (top panel: H&E stain, scale bar 50 μm). The immunofluorescence with two antibodies against glycosylated α-DG reveals a complete loss of functional α-DG glycosylation. Antibodies against dystrophin, α-DG core protein, β-DG and laminin α2 show mildly reduced to normal staining (scale bar 100 μm). (c) Western blot of skeletal muscle biopsy (MB) and skin fibroblasts (Fib) of control and ISPD-WWS P1. Both patient samples reveal core α-DG hypoglycosylation and loss of α-DG functional glycosylation. Glycoproteins were WGA-enriched from muscle or cell lysates (MB: 250μg protein/lane, Fib: 1000μg/lane). The immunoblot was probed with antibodies against the glycosylated form of α-DG (IIH6) and core α-DG (G6317), as well as withβ-DG (AP83) as a loading control.

Our first step toward identifying the underlying genetic defect in the large complementation cohort, was to perform linkage analysis to identify candidate regions. As reliable family history regarding consanguinity was not available for all cases, regions of homozygosity-by-descent were identified using high-resolution SNP arrays. Besides the sibling pair P2 and P3, four of the five unrelated patients showed multiple long (>10cM) stretches of homozygosity, suggesting some degree of consanguinity (Supplementary Fig. 3). We searched for regions where P2 and P3 are identical on both alleles and all or a subset of the four suspected consanguineous samples are homozygous. All 7,113 coding exons across 14 overlapping intervals were subjected to targeted sequencing (Supplementary Table 3). All seven samples were bar-coded, pooled, captured by one custom-designed capture array and sequenced on a lane of an Illumina HiSeq2000 flowcell as a 50bp paired-end run. The sequence data were processed through a custom-built analysis pipeline, and after variant filter strategies were applied, six genes were identified for which at least two independent protein-damaging variants passed the hard-filtration (Supplementary Table 4). Based on genetic evidence, ISPD was the most likely candidate gene. After manually examining the variants that did not meet filter criteria, as well as augmentation of the dataset with Sanger sequencing of P5 which was poorly covered, a total of four heterozygous variants and four homozygous variants were found in ISPD which identified multiple rare variants in all six independent cases (Table 1, Supplementary Fig. 4). All mutations were predicted to damage or abolish protein function, as expected in individuals with a severe form of dystroglycanopathy such as WWS[7]. In addition, ISPD was localized to chromosome 7p21.2, a region in which three of the four suspected consanguineous patients had intervals of homozygosity longer than 10cM and P2 and P3 shared both parental alleles (Fig. 4a,b). A schematic representation of all ISPD mutations identified in our patient cohort is shown in Fig. 4c.

Table 1

A summary of pathogenic ISPD mutations detected in this study

Patients	Zygosity	Chr	Genomic position (b37)	Nucleotide variant	Amino acid	Comment
P1	heterozygous	7	16,415,758	c.643C>T	p.Gln215*	nonsense mutation
	heterozygous	7	g.(16,107,358–16,115,680)_(16,289,931–16,297,326)del			exon 9–10 deletion
P2 and P3 (siblings)	heterozygous	7	16,348,146	c.789+2T>G	IVS4+2T>G	ivs4 splice-site mutation, ΔExon4
	heterozygous	7	16,445,940	c.277–279del ATT	p.Ile93del	single amino-acid (aa) deletion
P4	homozygous	7	16,131,322	c.1354T>A	p.*452Arg	mutation of original stop-codon, next stop codon 27 aa downstream
P5	homozygous	7	16,255,823	c.1120-1G>T	IVS8-1G>T	ivs8 splice site mutation, ΔExon9
P6	homozygous	7	g.(16,401,191–16,406,273)_(16,409,318–16,431,594)del			in frame deletion of Exon 3
P7	homozygous	7	16,415,851	c.550C>T	p.Arg184*	nonsense mutation

Fig. 4

Identification and validation of ISPD as disease gene in WWS patients

(a) Alignment of identical-by-descent (IBD) and homozygosity-by-descent (HBD) intervals among ISPD patients on chromosome 7 is shown with the genomic position in hg19 coordinates on the top and chromosome bands at the bottom. The minimal region of overlap between the three out of four suspected consanguineous samples were homozygous where P2 and P3 share both parental alleles is highlighted by a red box and (b) zoomed in to show the genes within the region. (c) Schematic representation (not drawn to scale) of the ISPD exon-intron gene structure. Human ISPD cDNA (NM_001101426, 5,524 bp) contains 10 coding exons spread across 333,796 bp genomic DNA. All identified pathogenic ISPD protein changes are indicated, as are regions with gene deletions and splice-site mutations. Coding exons are indicated by black boxes, and untranslated regions (UTR) by open boxes. (d) On-Cell western-based complementation assay of control and ISPD-WWS patient fibroblasts after nucleofection with a wild-type or mutant ISPD expression construct. Rescue of α-DG functional glycosylation was detected with α-DG glyco (IIH6) antibodies. (e) Adenovirus-mediated ISPD gene transfer rescues α-DG glycosylation defect in ISPD-WWS P2 patient cells. WGA-enriched cell lysates from fibroblasts were subjected to immunoblotting with α-DG glyco (IIH6), α-DG core (G6317), anti-myc (4A6), and β-DG (AP83) and by laminin overlay. Infection with ISPD-myc expressing adenovirus restored functional glycosylation in ISPD-WWS P2 patient cells, but did not significantly alter α-DG functional glycosylation in control cells.

To confirm the pathogenicity of the identified ISPD mutations, we conducted complementation assays on fibroblasts derived from the ISPD-WWS patients. In the patient cells, expression of wild-type ISPD, but not that of a mutant isoform (P6, ISPD-ΔE3) restored functional glycosylation (Fig. 4d); in control cells, such overexpression did not significantly alter functional α-DG glycosylation (Fig. 4e). Functional rescue of patient cells confirmed that the identified ISPD mutations have pathogenic relevance, and indicated that severe mutations in ISPD can cause WWS.

Notably, ISPD has not been characterized in mammals. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that ISPD is ubiquitously expressed in all tissues analyzed, with expression highest in brain (Supplementary Fig. 5). ISPD belongs to the family of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) synthases (also known as 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferases), that are conserved from bacteria to mammals (Supplementary Fig. 6). In E.coli, IspD activity contributes to the methylerythritol pathway (MEP) in its synthesis of isoprenoid precursors[16,17], which give rise to the polyisoprenoid alcohols (e.g., dolichols and polyprenols) found in all living organisms[18] (Supplementary Fig. 7). However, the MEP pathway is used only by eubacteria, green algae and the chloroplasts of higher plants[19]. Eukaryotes, archaebacteria, and the cytosol of higher plants are thought to use an alternative, ISPD-independent mevalonate (MVA) pathway for isoprenoid synthesis[20]. As the MEP pathway involving ISPD is postulated to be absent from animals, the specific role of ISPD in humans, especially in regards to α-DG glycosylation, is unclear.

To investigate the role of ISPD in α-DG glycosylation, we tested ISPD-WWS patients for changes in any known steps in laminin binding glycan synthesis. Notably, protein O-mannosylation, the initiating step, was markedly reduced in fibroblasts lacking functional ISPD, as were downstream events like O-mannosyl phosphorylation and LARGE-induced hyperglycosylation (Fig. 5a–c). These findings suggest that ISPD function is crucial for efficient POMT-dependent O-mannosylation and subsequent glycosylation of α-DG (Supplementary Fig. 8).

Fig. 5

ISPD loss of function causes α-DG O-mannosylation defect

(a) POMT activity in control cells and patient-derived dermal fibroblasts, as assayed by the rate of radioactive [3H]-mannose transfer from Dol-P-[3H]-Man (125,000 dpm/pmol) to a GST-α-DG fusion protein. POMT1-WWS and ISPD-WWS patient cells show comparable defects in POMT enzyme activity. Specific POMT activity in control cells was determined as 536.7 pmol/g/h. The diagram shows relative POMT activity in reference to control cells (n=3). Error bars represent s.d. (b) [32P]-orthophosphate labeling of DGFc5-expressing cultured cells from control and ISPD P2 cells. After O-mannosyl residues are transferred, the radioactive [32P]-orthophosphate is incorporated to form a phosphorylated O-linked mannose glycan[21]. The ISPD sample shows markedly reduced [32P] labeling due to reduced number of O-mannosyl acceptor sites. (c) Quantitative On-Cell western analysis of LARGE-induced α-DG hyperglycosylation. The glycosyltransferase LARGE participates in a post-phosphoryl modification transferring the laminin-binding glycan. Forced expression of LARGE increases the affinity of the cell surface for the IIH6 antibody in control cells, but not in POMT1 and ISPD deficient WWS cells, confirming that the mutant cells lack the O-mannosyl acceptors of the post-phosphoryl modification. IIH6 On-Cell quantitative data were normalized with DRAQ5 cell DNA dye (n=3). Error bars represent s.d.

In this study, we have identified a novel WWS disease gene in patient fibroblasts by using a complementation assay in combination with targeted sequencing. This approach provided conclusive genetic and biochemical evidence that recessive mutations in ISPD lead to impaired α-DG O-mannosylation, establishing a novel WWS pathomechanism. Further studies are needed to determine how defects in ISPD influence protein O-mannosylation, as this is the first WWS gene without proposed glycosyltransferase activity and direct role in α-DG glycosylation.

METHODS

Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/.