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Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant α-dystroglycan (αDG) glycosylation. Here, we report mutations in the isoprenoid synthase domain-containing (ISPD) gene as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but its role in chordates has been obscure to date because this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated αDG. These results implicate a role for ISPD in αDG glycosylation to maintain sarcolemma integrity in vertebrates.
Defective O-linked glycosylation of αDG is the characteristic feature of a clinically and genetically heterogeneous group of disorders, commonly referred to as dystroglycanopathies. This group of diseases is characterized by a broad phenotypic spectrum ranging from severe forms of CMD with eye involvement, cerebral malformations and intellectual disability including WWS and Muscle Eye Brain disease (MEB), to milder adult-onset phenotypes without central nervous system involvement such as limb girdle muscular dystrophy (LGMD) type 2I1. Mutations in six genes, POMT1, POMT2, POMGnT1, FKTN, FKRP and LARGE, encoding proteins involved in the post-translational modification of αDG, have been implicated in WWS and other dystroglycanopathies1-10. Mutations in these six genes represent 35% of WWS incidence, suggesting that additional WWS genes await discovery. At the milder end of the dystroglycanopathy disease spectrum single patients have been reported with αDG hypoglycosylation due to a missense mutation in either the dystroglycan gene (DAG1)11 or the DPM3 gene12, the latter leading to reduced levels of dolichol-P-mannose donor for O-mannosylation12. The factors determining dystroglycanopathy phenotypes are not well understood, but may involve the extent of residual glycosylation of αDG1,13,14 and other proteins15-17. Identifying novel causative genes will shed light on the pathological mechanisms of WWS and other dystroglycanopathies.
To identify novel genes that are involved in WWS, we selected a cohort of 59 patients with idiopathic WWS in whom mutations in the known dystroglycanopathy genes had been previously excluded. Thirty of these patients, the majority of whom came from consanguineous families, were genotyped using the Affymetrix GeneChip Human Mapping 250K SNP NspI Array to identify copy number variants (CNVs) and homozygous regions. SNP haplotyping detected a large number of non-overlapping homozygous regions amongst these patients, providing support for further genetic heterogeneity in WWS (Supplementary Fig. 1). Interestingly, the corresponding CNV profiles identified two homozygous deletions affecting the ISPD gene (patients WWS-160 and WWS-161, Supplementary Fig. 2), suggesting that ISPD is a strong candidate for WWS, as there were no other overlapping CNV regions present in the patient cohort. Another family (WWS-25; for pedigree see Supplementary Fig. 3a) was identified with two siblings and a cousin affected with WWS who shared a 3.5 Mb homozygous region on chromosome 7p21 containing ten genes including ISPD (Fig. 1a). One of these siblings was investigated by exome sequencing and after filtering based on an autosomal recessive pattern of inheritance according to methods described previously18, a single homozygous variant was identified in this region of shared homozygosity. This c.647C>A transversion in exon 3 of ISPD (NM_001101426.3), predicting a p.Ala216Asp substitution, showed complete segregation in the family, being present in the homozygous state in the other two affected individuals, in the heterozygous state in both parental couples and absent in healthy or deceased siblings with another phenotype.
Next, we sequenced the ten coding exons of ISPD in the WWS patient cohort and identified missense, nonsense and frameshift mutations in an additional five individuals from five families (Supplementary Fig. 3). In addition, failure to PCR-amplify exons 3 to 5 in family WWS-37, suggested the presence of a homozygous intragenic microdeletion, which was confirmed by quantitative MLPA analysis (data not shown). An overview of all identified mutations is given in Table 1 and a schematic representation of their localization is shown in Figure 1a. In summary, mutations affecting ISPD were detected in 9 out of 94 families, accounting for an overall percentage of 10% (Fig. 1b). All mutations showed a segregation pattern expected for causative recessive mutations in family members available for testing and were absent in a control cohort of 3712 haploid genomes. The three nonsense mutations, p.Arg268*, p.Lys278* and p.Glu396*, are predicted to give rise to nonsense-mediated mRNA decay or truncation of the protein. The three missense mutations (p.Ala216Asp, p.Arg126His and p.Ala122Pro) are located within the ISPD domain (Fig. 1a; Supplementary Fig. 4a), a conserved domain of a large GT-A glycosyltransferase family that also includes nucleotidyltransferases19. Both p.Ala216Asp and p.Arg126His affect highly conserved amino acids and are predicted to be damaging by PolyPhen2, whereas p.Ala122Pro is predicted to be probably damaging. In silico modeling based on an E. coli IspD crystal structure using the HOPE web server (see URLs) predicted charge and size differences between the wild-type and mutant amino acids that are likely to affect protein folding and disruption of the CTP-binding pocket for mutations p.Ala122Pro and p.Arg126His (Supplementary Fig. 4b).
All affected individuals with ISPD mutations had a severe WWS-like phenotype with only two out of 11 surviving beyond two years of age with brain anomalies that are more indicative of MEB (WWS-81 and -163; Supplementary Table 1). Routine cerebral MRI (Fig. 2a and 2b) showed typical features of cobblestone lissencephaly together with hydrocephalus, cerebellar hypoplasia and a kinked brainstem. Muscle histology and immunohistochemistry showed dystrophic changes and clear reduction of glycosylated αDG by the IIH6 antibody, which recognizes an unknown glyco-epitope on αDG20 (patients from families WWS-25, -160, and -81) (Fig. 2c-f).
The function of ISPD in vertebrates is unknown. In view of the significant conservation of protein sequences (65% amino acid similarity) between the zebrafish (Danio rerio) and human orthologs, we determined the effects of loss of function of zebrafish ispd, which encodes two isoforms that differ only in their N-termini. To do this, we knocked down both ispd isoforms with antisense morpholino oligonucleotides (MO), targeting exon-intron splice sites common to both transcripts (Supplementary Fig. 5). High doses of ispd MO1 (7 ng) caused hydrocephalus and incomplete brain folding in 82% of embryos (n=88) by 48 hours post fertilization (h.p.f.) (Fig. 3a and Supplementary Fig. 6), as well as significantly reduced eye size reminiscent of microphthalmia in WWS patients (Fig. 3b and 3c). Other phenotypic features included impaired motility and myotome lesions (data not shown). Injection of ispd MO2 (3 ng) caused similar morphological abnormalities, assuring the specificity of both MOs (Supplementary Fig. 7). We looked for structural defects in muscle fibers by labeling sarcolemma with membrane-localized red fluorescent protein (mRFP) and filamentous-actin (F-actin) with phalloidin. ispd MO1-injected embryos showed muscle fiber degeneration by 72 h.p.f. (Fig. 3d) and, in some cases, disruption of myotendinous junctions (MTJ; 45%, n=31), exemplified by elongated muscle fibers spanning MTJ (Fig. 3d). Together, these results suggest that zebrafish ispd knockdown embryos recapitulate the major aspects of human WWS pathology.
Hypoglycosylation of αDG is a diagnostic characteristic of WWS. To test if knockdown of ispd in zebrafish also affects glycosylation of αDG, we performed western blotting with the IIH6 antibody20. Compared with control embryos, glycosylated αDG was reduced in ispd MO1-injected zebrafish embryos (Fig. 4a). Given the similarity of MTJ disruption observed in ispd MO1-injected embryos and zebrafish laminin mutants21,22, the localization of laminins was assessed. Despite the severe muscle fiber degeneration, laminins remained localized to the MTJ in ispd MO1-injected embryos (Fig. 4b). Subsequently, we assessed the sarcolemma integrity in embryos injected with either ispd MO using Evans blue dye (EBD). Intact sarcolemma is impermeable to EBD. We found that MTJ-anchored muscle fibers were infiltrated by EBD before the onset of muscle degeneration at 48 h.p.f. (Fig. 4c and Supplementary Fig. 7c). Muscle pathology became evident as EBD-infiltrated muscle fibers retracted during muscle degeneration. The posterior myotome of ispd MO1-injected embryos was more susceptible to sarcolemma damage than the anterior myotome (Fig. 4c). As sarcolemma damage was reported in dystrophin-deficient models23,24, we assessed the immunoreactivity of dystrophin in ispd MO1-injected embryos. No obvious alterations to dystrophin immunoreactivity were detected (data not shown). Together, these results demonstrate an important WWS pathogenic mechanism, independent from laminin and dystrophin, in which loss of Ispd function in zebrafish results in αDG hypoglycosylation and compromised sarcolemma integrity, preceding muscle fiber degeneration.
In plants, protozoa and some bacteria, ISPD belongs to the non-mevalonate isoprenoid biosynthesis (MEP) pathway, which is absent in vertebrates25. Prokaryotic IspD has cytidyltransferase activity using 2-C-methyl-D erythritol as substrate for the synthesis of the nucleotide sugar CDP-methyl-erythritol. The structurally homologous TarI in Streptococcus pneumoniae, lacking the MEP pathway, uses ribitol-1-phosphate to produce an activated nucleotide sugar (CDP-ribitol) used for incorporation in polysaccharides26. Analogous to the role of DPM3, it is likely that human ISPD synthesises a novel nucleotide sugar, the exact nature of which remains to be determined. Glycosyltransferases could use such nucleotide-activated building blocks for incorporation into the αDG O-mannosyl glycan. Intriguingly, the bacterium Prevotella tannerae expresses a gene (accession number ZP_05736246) with both IspD and LicD (lipopolysaccharide core D) domains, which may act sequentially in the post-translational modifications. The LicD domain is also found in the putative glycosyltransferases, FKTN and FKRP, both involved in the glycosylation of αDG. Thus, it appears that the ISPD and LicD domains that are both contained in a prokaryotic precursor protein are dispersed over the vertebrate ISPD and FKTN/FKRP proteins, respectively. We sought to test the possibility of genetic interactions between these genes. Co-injection of sub-effective doses of ispd MO1 with fktn or fkrp MO showed a marked increase in the proportion of embryos with hydrocephalus (68% and 49% respectively, compared with 3% in control MO and ispd MO1 co-injection; Fig. 4d and Supplementary Fig. 8a). The same effect was seen using ispd MO2, confirming specificity (Supplementary Fig. 8). Moreover, glycosylation of αDG is reduced in the embryos co-injected with ispd MO1 and fktn/fkrp MO as compared to embryos co-injected with ispd MO1 and control MO, and single fktn/fkrp MO-injected embryos (Fig. 4e). These results support a cooperative interaction between ispd and fktn/fkrp in αDG glycosylation.
In conclusion, our findings provide evidence for a significant contribution of ISPD mutations to the prevalence of WWS. We report the identification of ISPD mutations in nine WWS families. Due to the high frequency of ISPD mutations in this WWS cohort (15% of pre-screened patients, 10% overall, Fig. 1b), we recommend that ISPD mutation analysis should be performed as part of routine molecular diagnostic testing in WWS. With the identification of ISPD, we can now explain almost 50% of all WWS of our cohort. The results of homozygosity mapping indicate the existence of several additional loci. Given that most of the remaining patients represent isolated cases, we anticipate that exome sequencing will be the strategy of choice to resolve additional genes and to unravel the complex post-translational modification pathways that are key to normal brain and muscular development.
We thank all family members who participated in this study. We would also like to thank A. Charon, Service de Pédiatrie, Grand Hôpital de Charleroi, Charleroi, Belgium for referring an affected patient, G. Powell and S. Gerety for critical comments on the manuscript and R. Schot for study of the deletion in family WWS-161. This project was supported by the Large-Scale Integrating Project GENCODYS-Genetic and Epigenetic Networks in Cognitive DYSfunction (241995), which is funded by the EU FP7 Health program (HvB), the Australian NHMRC with an overseas post-doctoral fellowship (TR), The Prinses Beatrix Fund (grant W.OR09-15 to DL and HvB), the Hersenstichting Nederland (HvB), and an EMBO Long-Term Fellowship (ALTF 805-2009 to KB). All zebrafish work was sponsored by the Wellcome Trust [grant number WT 077047/Z/05/Z] and [grant number WT 077037/Z/05/Z]. NGS experiments were financially supported by the Department of Human Genetics, Nijmegen, The Netherlands, as well as by the Netherlands Organization for Health Research and Development (ZonMW grant 916-86-016 to LELMV).
URLs UCSC Genome Bioinformatics, http://www.genome.ucsc.edu/; OMIM and Unigene, http://www.ncbi.nlm.nih.gov/; Ensembl, http://www.ensembl.org/index.html; PolyPhen2 http://genetics.bwh.harvard.edu/pph2/; Project HOPE http://www.cmbi.ru.nl/hope/home; Exome Variant Server http://snp.gs.washington.edu/EVS/; ZFIN http://www.zfin.org/
AUTHOR CONTRIBUTIONS The study was designed and the results were interpreted by T.R., K.B., D.L.S., Y.Y.L., H.G.B., D.J.L. and H.V.B. Subject ascertainment and recruitment was carried out by I.M., U.A., B.G., G.M.S.M., P.D., M.A.W., D.P.R., D.C., C.B., E.S., E.A.J.P., G.M.B.T.S., C.E.D.D.S., K.D., H.K., O.A.E.F.E.H., and H.V.B. Sequencing, CNV analysis and genotyping was carried out and interpreted by T.R., E.J.K., K.B., I.M., J.V.R., C.V.D.E., E.V.B., M.R., R.P., L.E.L.M.V., M.S., M.F.B., H.Z., J.A.V., C.G., G.M.S.M., and H.V.B. Zebrafish studies were designed and carried out by K.B., D.L.S. and Y.Y.L. Bioinformatic data analysis and protein modeling was done by T.R., J.V.R., C.G., and D.J.L. The manuscript was drafted by T.R., K.B., D.J.L., Y.Y.L. and H.V.B. All authors contributed to the final version of the paper.
COMPETING FINANCIAL INTERESTS The authors declare that they have no competing financial interests.