Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominantnegative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.epistasis | ciliopathy | zebrafish | in vivo assays E xome and whole-genome resequencing is likely to catalyze a paradigm shift in the identification of genetic lesions in patients (1). At the same time, even within the confines of the coding genome, such technologies pose an interpretive problem, in that the pathogenic candidacy of mutations can only be derived by narrow genetic models and limited computational predictive tools, both of which are likely to under-and misinterpret the effect of some mutations. Moreover, inter-and intrafamilial variability, a phenomenon prevalent in most genetic traits, remains a major confounding factor because both allelic variation at a single locus and second-site trans modifiers can exert a significant influence on penetrance and expressivity through additive and epistatic effects (2).Bardet-Biedl Syndrome (BBS) is a useful model for dissecting epistasis because most of the 14 BBS genes can also contribute epistatic alleles (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). BBS is also a representative of the ciliopathy disease spectrum, a group of disorders characterized by defects in ciliary structure and/or ciliary signal output (18). Hallmarks of BBS include retinal degeneration, obesity, hypogonadism, polydactyly, renal dysfunction, and mental retardation (19).We and others have shown that the zebrafish provides experimentally tractable and physiologically relevant models of important aspects of ciliary dysfunction (2,15,17,(20)(21)(22)(23). Moreover, human mRNA for ciliopathy genes can rescue both morphant and mutant zebrafish phenotypes efficiently, providing a robust platform for interpretation of the pathological relevance of identified missense alleles, whose causal relation to the disorder cannot be proven definitively with genetic arguments alone (15,17,22,23).Here, we have integrated multiple independent in vivo assays, followed by in vitro validations, to int...

SummaryPrimary cilia are conserved organelles that play crucial roles as mechano-and chemosensors, as well as transducing signaling cascades. Consequently, ciliary dysfunction results in a broad range of phenotypes: the ciliopathies. Bardet-Biedl syndrome (BBS), a model ciliopathy, is caused by mutations in 16 known genes. However, the biochemical functions of the BBS proteins are not fully understood. Here we show that the BBS7 protein (localized in the centrosomes, basal bodies and cilia) probably has a nuclear role by virtue of the presence of a biologically confirmed nuclear export signal. Consistent with this observation, we show that BBS7 interacts physically with the polycomb group (PcG) member RNF2 and regulate its protein levels, probably through a proteasome-mediated mechanism. In addition, our data supports a similar role for other BBS proteins. Importantly, the interaction with this PcG member is biologically relevant because loss of BBS proteins leads to the aberrant expression of endogenous RNF2 targets in vivo, including several genes that are crucial for development and for cellular and tissue homeostasis. Our data indicate a hitherto unappreciated, direct role for the BBS proteins in transcriptional regulation and potentially expand the mechanistic spectrum that underpins the development of ciliary phenotypes in patients.

Ciliary dysfunction has emerged as a common factor underlying the pathogenesis of both syndromic and isolated kidney cystic disease, an observation that has contributed to the unification of human genetic disorders of the cilium, the ciliopathies. Such grouping is underscored by two major observations: the fact that genes encoding ciliary proteins can contribute causal and modifying mutations across several clinically discrete ciliopathies, and the emerging realization that an understanding of the clinical pathology of one ciliopathy can provide valuable insight into the pathomechanism of renal cyst formation elsewhere in the ciliopathy spectrum. In this review, we discuss and attempt to stratify the different lines of proposed cilia-driven mechanisms for cystogenesis, ranging from mechano- and chemo-sensation, to cell shape and polarization, to the transduction of a variety of signaling cascades. We evaluate both common trends and differences across the models and discuss how each proposed mechanism can contribute to the development of novel therapeutic paradigms.

CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet-Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo, at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.

Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR also presents in various syndromes, including Shah-Waardenburg syndrome (WS), Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.Bardet-Biedl ͉ neural crest cells ͉ genetic interaction ͉ zebrafish H irschsprung disease (HSCR, MIM 164761) is the most common (1/5,000 live births) form of structural intestinal obstruction. It is defined by the absence of neural crest (NC)-derived enteric ganglia along a variable length of the bowel, invariably involving the recto-anal junction. This phenotype has been attributed to defects in migration, proliferation, and/or survival of the NC cells (NCC) that normally give rise to all neurons and supporting cells of the enteric nervous system (ENS), defining HSCR as a neurocristopathy. HSCR is also a useful model oligogenic disorder; it displays non-Mendelian modes of inheritance with low, sex-dependant penetrance in isolated HSCR cases. Although oligogenic multiplicative models have been proposed, mutations in the RET proto-oncogene have emerged as pivotal (1-4). Almost all HSCR patients harbor either a heterozygous mutation in the RET coding sequence or, more frequently, a hypomorphic allele in a conserved noncoding element in intron 1 that acts as a spatially restricted transcriptional enhancer (4-6).In addition to HCSR, some 30% of patients also exhibit other congenital anomalies as the result of chromosomal rearrangements [mostly Down syndrome (DS)], monogenic Mendelian disorders regardless of the mode of inheritance, or undiagnosed associations (6). In such cases, penetrance for the HSCR trait is 5 to 70%, suggesting additional predisposing genetic factor(s). Interestingly, alleles at the RET locus can have a role in modifying the risk of HSCR to be associated with several HSCR predisposing syndromes [congenital central hypoventilation, DS, Waardenburg (WS) type IV due to EDNRB mutations, and Bardet-Biedl (BBS)], but not all (Mowat-Wilson and Waardenburg type IV due to SOX10 mutations) (6-8). These observations are suggestive of RET-dependent and RET-independent HSCR cases (9); consistent with RET acting as a modifier gene in some HSCR predisposing syndromes, no correlation between the genotype for the syndrome disease causing gene and the HSCR trait could be drawn in monogenic HSCR syndromes (9,10). Surprisingly, the greatest RET ...

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