J. van Reeuwijk

POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker-Warburg syndrome

Background: Walker-Warburg syndrome (WWS) is an autosomal recessive condition characterised by congenital muscular dystrophy, structural brain defects, and eye malformations. Typical brain abnormalities are hydrocephalus, lissencephaly, agenesis of the corpus callosum, fusion of the hemispheres, cerebellar hypoplasia, and neuronal overmigration, which causes a cobblestone cortex. Ocular abnormalities include cataract, microphthalmia, buphthalmos, and Peters anomaly. WWS patients show defective O-glycosylation of α-dystroglycan (α-DG), which plays a key role in bridging the cytoskeleton of muscle and CNS cells with extracellular matrix proteins, important for muscle integrity and neuronal migration. In 20% of the WWS patients, hypoglycosylation results from mutations in either the protein O-mannosyltransferase 1 (POMT1), fukutin, or fukutin related protein (FKRP) genes. The other genes for this highly heterogeneous disorder remain to be identified. Objective: To look for mutations in POMT2 as a cause of WWS, as both POMT1 and POMT2 are required to achieve protein O-mannosyltransferase activity. Methods: A candidate gene approach combined with homozygosity mapping. Results: Homozygosity was found for the POMT2 locus at 14q24.3 in four of 11 consanguineous WWS families. Homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Immunohistochemistry in muscle showed severely reduced levels of glycosylated α-DG, which is consistent with the postulated role for POMT2 in the O-mannosylation pathway. Conclusions: A fourth causative gene for WWS was uncovered. These genes account for approximately one third of the WWS cases. Several more genes are anticipated, which are likely to play a role in glycosylation of α-DG.

Glyc-O-genetics of Walker-Warburg syndrome

Walker–Warburg syndrome (WWS) is the most severe of a group of multiple congenital anomaly disorders known as the cobblestone lissencephalies. These are characterized by congenital muscular dystrophy in conjunction with severe brain malformation and ocular abnormalities. In the last 3 years, important progress has been made towards the elucidation of the genetic causes of these disorders. Mutations in three genes, POMT1, fukutin and FKRP, have been described for WWS, which together account for approximately 20% of patients with Walker–Warburg. It has become evident that some of the underlying genes may cause a broad spectrum of phenotypes, ranging from limb girdle muscular dystrophy type 2I to WWS. In some cases, a genotype–phenotype correlation can be recognized. In line with the known or proposed functions of the resolved genes, all patients with cobblestone lissencephaly show defects in the O‐linked glycosylation of the glycoprotein α‐dystroglycan. Perhaps, the missing genes underlying the remainder of the unexplained WWS patients have also to be sought in the pathways involved in O‐linked protein glycosylation.

POMT2 mutation in a patient with ‘MEB-like’ phenotype

Mutations in POMT2 have so far only been reported in patients with Walker-Warburg phenotype. We report heterozygous POMT2 mutations in an a girl with a milder phenotype characterized by mental retardation, microcephaly, hypertrophy of the quadriceps and calf muscles, and structural brain changes mostly affecting the posterior fossa. Our findings suggest that, as previously reported for POMT1 and FKRP, mutations in the POMT2 can also be associated with clinical heterogeneity.

A homozygous FKRP start codon mutation is associated with Walker-Warburg syndrome, the severe end of the clinical spectrum

van Reeuwijk J, Olderode‐Berends MJW, van den Elzen C, Brouwer OF, Roscioli T, van Pampus MG, Scheffer H, Brunner HG, van Bokhoven H, Hol FA. A homozygous FKRP start codon mutation is associated with Walker–Warburg syndrome, the severe end of the clinical spectrum.

Liver cyst gene knockout in cholangiocytes inhibits cilium formation and Wnt signaling

Mutations in the PRKCSH, SEC63 and LRP5 genes cause autosomal dominant polycystic liver disease (ADPLD). The proteins products of PRKCSH (alias GIIB) and SEC63 function in protein quality control and processing in the endoplasmic reticulum (ER), while LRP5 is implicated in Wnt/β-catenin signaling. To identify common denominators in the PLD pathogenesis, we mapped the PLD interactome by affinity proteomics, employing both HEK293T cells and H69 cholangiocytes. Identification of known complex members, such as glucosidase IIA (GIIA) for PRKCSH, and SEC61A1 and SEC61B for SEC63, confirmed the specificity of the analysis. GANAB, encoding GIIA, was very recently identified as an ADPLD gene. The presence of GIIA in the LRP5 complex pinpoints a potential functional connection with PRKCSH. Interestingly, all three PLD-associated protein complexes included filamin A (FLNA), a multifunctional protein described to play a role in ciliogenesis as well as canonical Wnt signalling. As ciliary dysfunction may also contribute to hereditary liver cyst formation, we evaluated the requirement of PRKCSH and SEC63 for ciliogenesis and Wnt signaling. By CRISPR/Cas9 induced knockdown of both ADPLD genes in HEK293T cells and H69 cholangiocytes, we identified that their depletion results in defective ciliogenesis. However, only H69 knockouts displayed reduced Wnt3a activation. Our results suggest that loss of PRKCSH and SEC63 leads to general defects in ciliogenesis, while quenching of the Wnt signaling cascade is cholangiocyte-restricted. Interactions of all three PLD-associated protein complexes with FLNA may mark a common link between the ADPLD proteins and the cystogenic processes driving this disease.

The lebercilin-like protein is embedded in a ciliary protein network and is preferentially expressed in motile cilia

Mutations in LCA5 are causative for Leber congenital amaurosis, a severe hereditary retinal dystrophy in humans. Lebercilin, encoded by LCA5, localizes to connecting cilia of photoreceptor cells in the retina and specifically interacts with the intraflagellar transport (IFT) machinery. Bioinformatic analysis has identified lebercilin-like protein, previously known as C21orf13, as a lebercilin homolog in humans. In this study, we have characterized the molecular properties of lebercilin-like protein by defining the lebercilin-like interactome and assessing its (sub)cellular localization in ciliated cells. We show that lebercilin-like protein is embedded in a ciliary protein network and specifically localizes at the basal body and ciliary axoneme of ciliated cells, like lebercilin. mRNA expression studies indicate that lebercilin-like protein is preferentially expressed in tissues featuring motile cilia and/or flagella. Based on these data and bioinformatic co-expression profiling, we suggest that LCA5L is a likely candidate gene for motile ciliopathies such as Primary Ciliary Dyskinesia (PCD).

Multidisciplinary nephrogenetic outpatient clinic combined with diagnostic exome sequencing for improved diagnostics and treatment

Single gene disorders are estimated to account for ~30% of children and ~10% of adult patients attending renal outpatient services. For mutation detection by exome sequencing, deep phenotyping, reverse phenotyping and family history information are important. A multidisciplinary nephrogenetic outpatient clinic for children and adult patients with (genetic) kidney diseases has been established by a team of (pediatric) nephrologists and a clinical geneticist in the Radboudumc. Clinical exome sequencing for a broad spectrum of isolated- and syndromic renal (ciliary) disorders has been developed. The approach consists of a two-tier analysis in which the first step is to screen for pathogenic variants in genes that are known to be mutated in renal diseases (170 genes) or (renal) ciliopathies (125 genes). If causative mutations are not identified in the first step, the complete exome data set can be analysed with informed consent. The first results with the renal disease gene panel in 35 unrelated patients with undiagnosed renal disease led to pathogenic mutations in the CC2D2A, CLCN5, NPHP1 and UMOD gene, detected in four cases and in six other cases likely pathogenic variants needed follow-up studies. Further analysis of the complete exome data set in 13 patients, revealed possible pathogenic mutations in two cases. While variant and copy number variation analysis in the rest of exome is expected to further increase diagnostic yield, we can already conclude that the combination of the multidisciplinary outpatient clinic with diagnostic exome sequencing provides a powerful tool for detecting causative mutations of renal disease.

PDZD7 connects the Usher protein complex to the intraflagellar transport machinery

Several Usher syndrome (USH)-associated proteins are known to localize to the connecting cilium of photoreceptor cells. The unconventional myosin MYO7A (USH1B) was long accepted as the transport molecule responsible for the ciliary localization of USH proteins. However, based on the typical location of several of the USH proteins along the ciliary axoneme, the involvement of the main ciliary trafficking machinery, intraflagellar transport (IFT), seems apparent. The USH-associated scaffold protein PDZD7 is known to interact with SANS, Usherin, GPR98 and Whirlin, all of which can be found in the connecting cilium. Here, we report that PDZD7 provides the physical link of the USH-protein network to IFT-complex members. Tandem affinity purification (TAP) studies revealed a potential interaction between PDZD7 and several IFT molecules, including IFT25 and IFT27. TAP analyses of the other USH proteins were negative for IFT proteins, suggesting that this interaction is unique for PDZD7. In addition, a dedicated yeast two-hybrid screen of 200 (predicted) ciliary proteins revealed an interaction between PDZD7 and IFT57. The interaction between PDZD7 and selected IFT subunits was substantiated by co-immunoprecipitations. In accordance with these results, mRFP-tagged PDZD7, expressed in ciliated hTERT-RPE1 cells, localizes not only at the basal body, but also at the axoneme of a subset of cells. Pending further validation of the interaction between PDZD7 and IFT-B proteins, these first results suggest PDZD7 as a functional connection between USH-proteins and the IFT machinery. Future studies should reveal whether PDZD7 is involved in IFT of USH proteins in vivo.

From proteomic data to networks: statistics and methods reveal ciliary protein interaction landscape

Objective: The assembly of protein interaction networks (PIN) is an important step to understand the biological function of proteins. Affinity purification coupled to mass spectrometry (AP-MS) has become the technique of choice for the assembly and analysis of PINs. However, most current studies, especially in human cells, are focused on specific biological systems (e.g. the cilium) resulting in datasets of a small to intermediate scale. In such cases, methods that developed for genome-scale datasets are of limited utility. We propose here a framework that is specifically designed for the analysis of incomplete proteomic data focused on ciliary function and ciliopathies.

A systems biology approach towards the prediction of ciliopathy mechanisms

Objective Understanding the molecular and cellular mechanisms of ciliopathies is vital for dissecting their pathogenesis, identifying appropriate therapeutic targets and designing effective treatments. Recent advances in DNA sequencing technology have provided a torrent of genetic data that can now be used to elucidate the genetic basis of human diseases. A consensus has emerged among biologists that to fully exploit the available data, they have to be correlated with additional research. This is especially important for rare-disease genetics, because of the small number of available patients.