The field of gene therapy has made significant strides over the last several decades toward the treatment of previously untreatable genetic disease. Gene therapy techniques have been aimed at ...mitigating disease features of recessive and dominant disorders, as well as several cancers and other diseases. While there have been numerous disease targets of gene therapy trials, only four therapies have reached FDA and/or EMA approval for clinical use. Gene correction using CRISPR-Cas9 is an extension of gene therapy that has received considerable attention in recent years and boasts many possible uses beyond classical gene therapy approaches. While there is significant therapeutic potential using gene therapy and gene correction strategies, a number of hurdles remain to be overcome before they become more common in clinical use, particularly with regards to safety and efficacy. As research progresses in this exciting field, it is likely that these therapies will become first-line treatments and will have tremendous positive impacts on the lives of patients with genetic disorders.
Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, ...we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To devise a comprehensive multiplatform genetic testing strategy for inherited retinal disease and to describe its performance in 1000 consecutive families seen by a single clinician.
Retrospective ...series.
One thousand consecutive families seen by a single clinician.
The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed, and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to 1 of 62 diagnostic categories, and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from 2 to nearly 900 000.
Sensitivity and false genotype rate.
Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 75% of these 104 genes have coding sequences small enough to be packaged efficiently into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families), whereas mutations in 80 genes caused disease in 5 or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next-generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole-exome sequencing of the remaining 424 families revealed mutations in an additional 182 families, and whole-genome sequencing of 4 of the remaining 242 families revealed 2 additional genotypes that were invisible by the other methods. Performing the testing in a clinically focused tiered fashion would be 6.1% more sensitive and 17.7% less expensive and would have a significantly lower average false genotype rate than using whole-exome sequencing to assess more than 300 genes in all patients (7.1% vs. 128%; P < 0.001).
Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.
Abstract
The BBSome is a protein complex consisting of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 that associates with intraflagellar transport complexes and specializes in ciliary ...trafficking. In primary cilia, ciliary entry requires the fully assembled BBSome as well as the small GTPase, ARL6 (BBS3). Retinal photoreceptors possess specialized cilia. In light of key structural and functional differences between primary and specialized cilia, we examined the principles of BBSome recruitment to photoreceptor cilia. We performed sucrose gradient fractionation using retinal lysates of Bbs2−/−, Bbs7−/−, Bbs8−/− and Bbs3−/− mice to determine the status of BBSome assembly, then determined localization of BBSome components using immunohistochemistry. Surprisingly, we found that a subcomplex of the BBSome containing at least BBS1, BBS5, BBS8 and BBS9 is recruited to cilia in the absence of BBS2 or BBS7. In contrast, a BBSome subcomplex consisting of BBS1, BBS2, BBS5, BBS7 and BBS9 is found in Bbs8−/− retinas and is denied ciliary entry in photoreceptor cells. In addition, the BBSome remains fully assembled in Bbs3−/− retinas and can be recruited to photoreceptor cilia in the absence of BBS3. We compared phenotypic severity of their retinal degeneration phenotypes. These findings demonstrate that unlike primary cilia, photoreceptor cilia admit a partially assembled BBSome meeting specific requirements. In addition, the recruitment of the BBSome to photoreceptor cilia does not require BBS3. These findings indicate that the ciliary entry of the BBSome is subjected to cell-specific regulation, particularly in cells with highly adapted forms of cilia such as photoreceptors.
The primary cilium is a highly specialized and evolutionary conserved organelle in eukaryotes that plays a significant role in cell signaling and trafficking. Over the past few decades tremendous ...progress has been made in understanding the physiology of cilia and the underlying pathomechanisms of various ciliopathies. Syndromic ciliopathies consist of a group of disorders caused by ciliary dysfunction or abnormal ciliogenesis. These disorders have multiorgan involvement in addition to retinal degeneration underscoring the ubiquitous distribution of primary cilia in different cell types. Genotype-phenotype correlation is often challenging due to the allelic heterogeneity and pleiotropy of these disorders. In this review, we discuss the clinical and genetic features of syndromic ciliopathies with a focus on Bardet-Biedl syndrome (BBS) as a representative disorder. We discuss the structure and function of primary cilia and their role in retinal photoreceptors. We describe the progress made thus far in understanding the functional and genetic characterization including expression quantitative trait locus (eQTL) analysis of BBS genes. In the future directions section, we discuss the emerging technologies, such as gene therapy, as well as anticipated challenges and their implications in therapeutic development for ciliopathies.
Insulin and its receptor are critical for the regulation of metabolic functions, but the mechanisms underlying insulin receptor (IR) trafficking to the plasma membrane are not well understood. Here, ...we show that Bardet Biedl Syndrome (BBS) proteins are necessary for IR localization to the cell surface. We demonstrate that the IR interacts physically with BBS proteins, and reducing the expression of BBS proteins perturbs IR expression in the cell surface. We show the consequence of disrupting BBS proteins for whole body insulin action and glucose metabolism using mice lacking different BBS genes. These findings demonstrate the importance of BBS proteins in underlying IR cell surface expression. Our data identify defects in trafficking and localization of the IR as a novel mechanism accounting for the insulin resistance commonly associated with human BBS. This is supported by the reduced surface expression of the IR in fibroblasts derived from patients bearing the M390R mutation in the BBS1 gene.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, ...male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD.
Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and ...Bardet-Biedl syndrome (BBS), which are collectively termed “ciliopathies.” Recent protein–protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein–protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D)-dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B–INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins.
Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. ...Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A 30-Mile-per-Hour Headwind Sheffield, Val C
JAMA : the journal of the American Medical Association,
07/2018, Letnik:
320, Številka:
1
Journal Article
Recenzirano
Sheffield shares his treatment of an infant who was extremely small for gestational age, and was suspected with lethal or severe debilitating chromosomal abnormality. Upon examining the infant, ...Sheffield was fairly confident of a diagnosis with a poor prognosis, but knowing that chromosome testing could clarify the situation, he called the cytogenetics laboratory on-call person and asked that the baby's karyotype analysis be set up immediately so that a result might be ready by Monday. As he subsequently pondered the case, it occurred to him that while caring for this family he did not once think about relative value units (RVUs) or value-based purchasing.
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