The Genetic Drivers of Juvenile, Young, and Early-Onset Parkinson's Disease in India.

BACKGROUND: Recent studies have advanced our understanding of the genetic drivers of Parkinson's disease (PD). Rare variants in more than 20 genes are considered causal for PD, and the latest PD genome-wide association study (GWAS) identified 90 independent risk loci. However, there remains a gap in our understanding of PD genetics outside of the European populations in which the vast majority of these studies were focused. OBJECTIVE: The aim was to identify genetic risk factors for PD in a South Asian population. METHODS: A total of 674 PD subjects predominantly with age of onset (AoO) ≤50 years (encompassing juvenile, young, or early-onset PD) were recruited from 10 specialty movement disorder centers across India over a 2-year period; 1376 control subjects were selected from the reference population GenomeAsia, Phase 2. We performed various case-only and case-control genetic analyses for PD diagnosis and AoO. RESULTS: A genome-wide significant signal for PD diagnosis was identified in the SNCA region, strongly colocalizing with SNCA region signal from European PD GWAS. PD cases with pathogenic mutations in PD genes exhibited, on average, lower PD polygenic risk scores than PD cases lacking any PD gene mutations. Gene burden studies of rare, predicted deleterious variants identified BSN, encoding the presynaptic protein Bassoon that has been previously associated with neurodegenerative disease. CONCLUSIONS: This study constitutes the largest genetic investigation of PD in a South Asian population to date. Future work should seek to expand sample numbers in this population to enable improved statistical power to detect PD genes in this understudied group. © 2023 Denali Therapeutics and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genome-Wide Polygenic Score Predicts Large Number of High Risk Individuals in Monogenic Undiagnosed Young Onset Parkinson's Disease Patients from India.

Parkinson's disease (PD) is a genetically heterogeneous neurodegenerative disease with poorly defined environmental influences. Genomic studies of PD patients have identified disease-relevant monogenic genes, rare variants of significance, and polygenic risk-associated variants. In this study, whole genome sequencing data from 90 young onset Parkinson's disease (YOPD) individuals are analyzed for both monogenic and polygenic risk. The genetic variant analysis identifies pathogenic/likely pathogenic variants in eight of the 90 individuals (8.8%). It includes large homozygous coding exon deletions in PRKN and SNV/InDels in VPS13C, PLA2G6, PINK1, SYNJ1, and GCH1. Eleven rare heterozygous GBA coding variants are also identified in 13 (14.4%) individuals. In 34 (56.6%) individuals, one or more variants of uncertain significance (VUS) in PD/PD-relevant genes are observed. Though YOPD patients with a prioritized pathogenic variant show a low polygenic risk score (PRS), patients with prioritized VUS or no significant rare variants show an increased PRS odds ratio for PD. This study suggests that both significant rare variants and polygenic risk from common variants together may contribute to the genesis of PD. Further validation using a larger cohort of patients will confirm the interplay between monogenic and polygenic variants and their use in routine genetic PD diagnosis and risk assessment.

Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

Elevated Serum Amino Acids Induce a Subpopulation of Alpha Cells to Initiate Pancreatic Neuroendocrine Tumor Formation.

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing ß cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.

Validation of a Genome-Wide Polygenic Score for Coronary Artery Disease in South Asians.

BACKGROUND: Genome-wide polygenic scores (GPS) integrate information from many common DNA variants into a single number. Because rates of coronary artery disease (CAD) are substantially higher among South Asians, a GPS to identify high-risk individuals may be particularly useful in this population. OBJECTIVES: This analysis used summary statistics from a prior genome-wide association study to derive a new GPSCAD for South Asians. METHODS: This GPSCAD was validated in 7,244 South Asian UK Biobank participants and tested in 491 individuals from a case-control study in Bangladesh. Next, a static ancestry and GPSCAD reference distribution was built using whole-genome sequencing from 1,522 Indian individuals, and a framework was tested for projecting individuals onto this static ancestry and GPSCAD reference distribution using 1,800 CAD cases and 1,163 control subjects newly recruited in India. RESULTS: The GPSCAD, containing 6,630,150 common DNA variants, had an odds ratio (OR) per SD of 1.58 in South Asian UK Biobank participants and 1.60 in the Bangladeshi study (p < 0.001 for each). Next, individuals of the Indian case-control study were projected onto static reference distributions, observing an OR/SD of 1.66 (p < 0.001). Compared with the middle quintile, risk for CAD was most pronounced for those in the top 5% of the GPSCAD distribution-ORs of 4.16, 2.46, and 3.22 in the South Asian UK Biobank, Bangladeshi, and Indian studies, respectively (p < 0.05 for each). CONCLUSIONS: The new GPSCAD has been developed and tested using 3 distinct South Asian studies, and provides a generalizable framework for ancestry-specific GPS assessment.

Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes.

BACKGROUND: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. METHODS: APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. RESULTS: APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. CONCLUSIONS: Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.

Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes.

BACKGROUND: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. METHODS: Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. RESULTS: Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. CONCLUSIONS: APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

Heterogeneity of Satellite Cells Implicates DELTA1/NOTCH2 Signaling in Self-Renewal.

How satellite cells and their progenitors balance differentiation and self-renewal to achieve sustainable tissue regeneration is not well understood. A major roadblock to understanding satellite cell fate decisions has been the difficulty of studying this process in vivo. By visualizing expression dynamics of myogenic transcription factors during early regeneration in vivo, we identify the time point at which cells undergo decisions to differentiate or self-renew. Single-cell RNA sequencing reveals heterogeneity of satellite cells, including a subpopulation enriched in Notch2 receptor expression, during both muscle homeostasis and regeneration. Furthermore, we reveal that differentiating cells express the Dll1 ligand. Using antagonistic antibodies, we demonstrate that the DLL1 and NOTCH2 signaling pair is required for satellite cell self-renewal. Thus, differentiating cells provide the self-renewing signal during regeneration, enabling proportional regeneration in response to injury while maintaining the satellite cell pool. These findings have implications for therapeutic control of muscle regeneration.

Evaluating the quality of the 1000 genomes project data.

BACKGROUND: Data from the 1000 Genomes project is quite often used as a reference for human genomic analysis. However, its accuracy needs to be assessed to understand the quality of predictions made using this reference. We present here an assessment of the genotyping, phasing, and imputation accuracy data in the 1000 Genomes project. We compare the phased haplotype calls from the 1000 Genomes project to experimentally phased haplotypes for 28 of the same individuals sequenced using the 10X Genomics platform. RESULTS: We observe that phasing and imputation for rare variants are unreliable, which likely reflects the limited sample size of the 1000 Genomes project data. Further, it appears that using a population specific reference panel does not improve the accuracy of imputation over using the entire 1000 Genomes data set as a reference panel. We also note that the error rates and trends depend on the choice of definition of error, and hence any error reporting needs to take these definitions into account. CONCLUSIONS: The quality of the 1000 Genomes data needs to be considered while using this database for further studies. This work presents an analysis that can be used for these assessments.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Next generation sequencing identifies novel disease-associated BEST1 mutations in Bestrophinopathy patients.

Bestinopathies are a spectrum of retinal disorders associated with mutations in BEST1 including autosomal recessive bestrophinopathy (ARB) and autosomal dominant Best vitelliform macular dystrophy (BVMD). We applied whole-exome sequencing on four unrelated Indian families comprising eight affected and twelve unaffected individuals. We identified five mutations in BEST1, including p.Tyr131Cys in family A, p.Arg150Pro in family B, p.Arg47His and p.Val216Ile in family C and p.Thr91Ile in family D. Among these, p.Tyr131Cys, p.Arg150Pro and p.Val216Ile have not been previously reported. Further, the inheritance pattern of BEST1 mutations in the families confirmed the diagnosis of ARB in probands in families A, B and C, while the inheritance of heterozygous BEST1 mutation in family D (p.Thr91Ile) was suggestive of BVMD. Interestingly, the ARB families A and B carry homozygous mutations while family C was a compound heterozygote with a mutation in an alternate BEST1 transcript isoform, highlighting a role for alternate BEST1 transcripts in bestrophinopathy. In the BVMD family D, the heterozygous BEST1 mutation found in the proband was also found in the asymptomatic parent, suggesting an incomplete penetrance and/or the presence of additional genetic modifiers. Our report expands the list of pathogenic BEST1 genotypes and the associated clinical diagnosis.

Comprehensive genomic analysis identifies pathogenic variants in maturity-onset diabetes of the young (MODY) patients in South India.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.

Whole exome sequencing reveals a functional mutation in the GAIN domain of the Bai2 receptor underlying a forward mutagenesis hyperactivity QTL.

The identification of novel genes underlying complex mouse behavioral traits remains an important step in understanding normal brain function and its dysfunction in mental health disorders. To identify dominant mutations that influence locomotor activity, we performed a mouse N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen and mapped several loci as quantitative traits. Here we describe the fine-mapping and positional cloning of a hyperactivity locus mapped to the medial portion of mouse chromosome four. We employed a modified recombinant progeny testing approach to fine-map the confidence interval from ≈20 Mb down to ≈5 Mb. Whole exome resequencing of all exons in this region revealed a single missense mutation in the adhesion G protein-coupled receptor brain-specific angiogenesis inhibitor 2 (Bai2). This mutation, R619W, is located in a critical extracellular domain that is a hotspot for mutations in this receptor class. We find that in two different mammalian cell lines, surface expression of Bai2 R619W is markedly reduced relative to wild-type Bai2, suggesting that R619W is a loss-of-function mutation. Our results highlight the powerful combination of ENU mutagenesis and next-generation sequencing to identify specific mutations that manifest as subtle behavioral phenotypes.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/ßKlotho Complex.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass.

Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and ß-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into ß-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels.

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain.

LDL (low-density lipoprotein) receptor (LDLR) binds to its negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9) through the first EGF (epidermal growth factor-like) domain [EGF(A)]. The isolated EGF(A) domain is a poor antagonist due to its low affinity for PCSK9. To improve binding affinity, we used a phage display approach by randomizing seven PCSK9 contact residues of EGF(A), including the Ca(2+)-coordinating Asp310. The library was panned in Ca(2+)-free solution, and 26 unique clones that bind to PCSK9 were identified. Four selected variants demonstrated improved inhibitory activities in a PCSK9-LDLR competition binding ELISA. The Fc fusion protein of variant EGF66 bound to PCSK9 with a K(d) value of 71 nM versus 935 nM of wild type [EGF(A)-Fc] and showed significantly improved potency in inhibiting LDLR degradation in vitro and in vivo. The five mutations in EGF66 could be modeled in the EGF(A) structure without perturbation of the EGF domain fold, and their contribution to affinity improvement could be rationalized. The most intriguing change was the substitution of the Ca(2+)-coordinating Asp310 by a Lys residue, whose side-chain amine may have functionally replaced Ca(2+). EGF66-Fc and other EGF variants having the Asp310Lys change bound to PCSK9 in a Ca(2+)-independent fashion. The findings indicate that randomization of an important Ca(2+)-chelating residue in conjunction with "selection pressure" applied by Ca(2+)-free phage selection conditions can yield variants with an alternatively stabilized Ca(2+) loop and with increased binding affinities. This approach may provide a new paradigm for the use of diversity libraries to improve affinities of members of the Ca(2+)-binding EGF domain subfamily.

Chondroitin sulfate synthase 1 (Chsy1) is required for bone development and digit patterning.

Joint and skeletal development is highly regulated by extracellular matrix (ECM) proteoglycans, of which chondroitin sulfate proteoglycans (CSPGs) are a major class. Despite the requirement of joint CSPGs for skeletal flexibility and structure, relatively little is understood regarding their role in establishing joint positioning or in modulating signaling and cell behavior during joint formation. Chondroitin sulfate synthase 1 (Chsy1) is one of a family of enzymes that catalyze the extension of chondroitin and dermatan sulfate glycosaminoglycans. Recently, human syndromic brachydactylies have been described to have loss-of-function mutations at the CHSY1 locus. In concordance with these observations, we demonstrate that mice lacking Chsy1, though viable, display chondrodysplasia and decreased bone density. Notably, Chsy1(-/-) mice show a profound limb patterning defect in which orthogonally shifted ectopic joints form in the distal digits. Associated with the digit-patterning defect is a shift in cell orientation and an imbalance in chondroitin sulfation. Our results place Chsy1 as an essential regulator of joint patterning and provide a mouse model of human brachydactylies caused by mutations in CHSY1.

Morphological defects in a novel Rdh10 mutant that has reduced retinoic acid biosynthesis and signaling.

Retinoic acid (RA) signaling is necessary for proper patterning and morphogenesis during embryonic development. Tissue-specific RA signaling requires precise spatial and temporal synthesis of RA from retinal by retinaldehyde dehydrogenases (Raldh) and the conversion of retinol to retinal by retinol dehydrogenases (Rdh) of the short-chain dehydrogenase/reducatase gene family (SDR). The SDR, retinol dehydrogenase 10 (RDH10), is a major contributor to retinal biosynthesis during mid-gestation. We have identified a missense mutation in the Rdh10 gene (Rdh10(m366Asp) ) using an N-ethyl-N-nitrosourea-induced forward genetic screen that result in reduced RA levels and signaling during embryonic development. Rdh10(m366Asp) mutant embryos have unique phenotypes, such as edema, a massive midline facial cleft, and neurogenesis defects in the forebrain, that will allow the identification of novel RA functions.

A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain.

Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia. The complex that we have described is essential for normal cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle.