Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects.

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering

The tuberous sclerosis proteins regulate formation of the primary cilium via a rapamycin-insensitive and polycystin 1-independent pathway

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome in which severe renal cystic disease can occur. Many renal cystic diseases, including autosomal dominant polycystic kidney disease (ADPKD), are associated with absence or dysfunction of the primary cilium. We report here that hamartin (TSC1) localizes to the basal body of the primary cilium, and that Tsc1−/− and Tsc2−/− mouse embryonic fibroblasts (MEFs) are significantly more likely to contain a primary cilium than wild-type controls. In addition, the cilia of Tsc1−/− and Tsc2−/− MEFs are 17–27% longer than cilia from wild-type MEFs. These data suggest a novel type of ciliary disruption in TSC, associated with enhanced cilia development. The TSC1 and TSC2 proteins function as a heterodimer to inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1). The enhanced ciliary formation in the Tsc1−/− and Tsc2−/− MEFs was not abrogated by rapamycin, which indicates a TORC1-independent mechanism. Polycystin 1 (PC1), the product of the PKD1 gene, has been found to interact with TSC2, but Pkd1−/− MEFs did not have enhanced ciliary formation. Furthermore, while activation of mTOR has been observed in renal cysts from ADPKD patients, Pkd1−/− MEFs did not have evidence of constitutive mTOR activation, thereby underscoring the independent functions of the TSC proteins and PC1 in regulation of primary cilia and mTOR. Our data link the TSC proteins with the primary cilium and reveal a novel phenotype of enhanced ciliary formation in a cyst-associated disease

Primary ciliary dyskinesia: current state of the art

Primary ciliary dyskinesia (PCD) is usually inherited as an autosomal recessive, and in classical form presents with upper and lower respiratory tract infection, and mirror image arrangement in around 50% cases. It is becoming appreciated that dysfunction of cilia is implicated in a wider spectrum of disease, including polycystic liver and kidney disease, central nervous system problems including retinopathy and hydrocephalus, and biliary atresia. Cilia are complex structures, containing more than 250 proteins, but recent studies have begun to locate PCD genes scattered throughout the genome. Screening tests for PCD include nasal nitric oxide and in vivo tests of ciliary motility such as the saccharin test. Specific diagnosis requires examination of cilia by light and electron microscopy, with epithelial culture in doubtful cases. This is only available in supra-regional centres, which have recently been centrally funded by NCG in order to provide a comprehensive, free diagnostic service in England. Treatment is unfortunately not evidence based, since there are no randomised controlled clinical trials in this condition, and recommendations are largely extrapolated from cystic fibrosis and other suppurative lung diseases

Designing a Parking Environment Accessible for Adult Tricycles in Lyon

Lyon, France is rapidly expanding its cycling infrastructure, yet it often overlooks the needs of individuals who use adaptive tricycles. In collaboration with La Ville à Vélo, we were tasked with designing a parking environment, which includes both the bicycle rack and the surrounding area, that is accessible to people who use tricycles. To inform our design, we conducted a comprehensive observation study to assess the accessibility of existing bike racks, alongside interviews with tricycle users, rack designers, and a Métropole official who all provided valuable inputs for our final design. The result is a thoughtfully designed parking system that enhances accessibility while aligning with the goals of the Métropole