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

Abstract

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 α-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.

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Figure 1: Status of α-dystroglycan glycosylation in dermal fibroblasts from subjects with dystroglycanopathy.
Figure 2: Cell fusion experiments reveal new genetic complementation groups.
Figure 3: Clinical presentation and α-dystroglycan glycosylation defect in ISPD-deficient subject P1 with WWS.
Figure 4: Identification and validation of ISPD as a disease-associated gene in subjects with WWS.
Figure 5: ISPD loss-of-function mutations cause α-dystroglycan O-mannosylation defect.

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Acknowledgements

We thank the Gene Transfer Vector Core (University of Iowa, supported by the US National Institutes of Health (NIH) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (P30 DK 54759) for generating adenoviruses, T. Toy at the UCLA Clinical Genomics Center (see URLs) for assisting with construction of the sequencing libraries, S. Feng at the UCLA Broad Stem Cell Research Center (BSCRC) for assisting in running HiSeq2000 and B. Harry at the UCLA Clinical Genomics Center for maintaining the data analysis pipeline. Sequencing and sequence analysis were supported by the Center for Duchenne Muscular Dystrophy and the UCLA Muscular Dystrophy Core Center grant from the NIH (5P30AR05723). This study used fibroblast samples from the National Institute of Neurological Disorders and Stroke (NINDS) Cell Line Repository (see URLs) and the Miami Brain and Tissue Bank for Developmental Disorders, funded by the National Commission for Human Development (NICHD). We thank members of the Campbell laboratory and C.A. Campbell for fruitful discussions, A. Dietz, A. Crimmins, G. Morgensen, J. Eskuri, P. Guicheney and H.v. Bokhoven for technical support and C. Blaumueller for critical reading of the manuscript. This work was supported in part by a Paul D. Wellstone Muscular Dystrophy Cooperative Research Center Grant (1U54NS053672 to K.P.C., K.D.M., S.A.M. and T.W.) and an American Recovery and Reinvestment Act (ARRA) Go Grant (1 RC2 NS069521-01 to K.P.C. and T.W.). The Muscular Dystrophy Campaign Grant to F.M. is also gratefully acknowledged. S.C. and F.M. are investigators of and are supported by the European Framework Programme 7 (FP7) NMD-Chip and Bio-NMD projects. F.M. is supported by the Great Ormond Street Hospital Children's Charity. K.P.C. is an investigator of the Howard Hughes Medical Institute.

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Authors

Contributions

T.W., H.L., S.F.N. and K.P.C. designed the research. T.W. performed the research and analyzed the data. H.L. and S.F.N. performed SNP analysis, next-generation sequencing and data filtering. M.L. and S.S. carried out POMT enzyme activity assays. T.Y.-M. performed α-dystroglycan orthophosphate cell labeling experiments. D.B.V.d.B. performed qRT-PCR expression analysis. D.V. carried out antibody affinity purification and labeling. T.L.W. carried out Sanger sequencing of known WWS-associated genes. S.A.M. performed muscle histology and clinical data interpretation. H.S., J.V., S.C., F.M., T.V., A.S.L., W.B.D. and K.D.M. provided clinical data and/or fibroblast samples from individuals with WWS. K.P.C. supervised and mentored the project. T.W. and K.P.C. wrote the initial manuscript, and all authors approved and commented on the manuscript.

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Correspondence to Kevin P Campbell.

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Willer, T., Lee, H., Lommel, M. et al. ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome. Nat Genet 44, 575–580 (2012). https://doi.org/10.1038/ng.2252

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