CRISPR-Cas9–based treatment of myocilin-associated glaucoma

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-of-function mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9–med iated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease. Keywords: myocilin; CRISPR; glaucoma; trabecular meshwork; genome editingNational Institutes of Health (U.S.) (Grant R01 EY024259)National Institutes of Health (U.S.) (Grant R01 EY026177)National Institutes of Health (U.S.) (Grant R00 EY022077

Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1

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

Cortical enlargement in autism is associated with a functional VNTR in the monoamine oxidase A gene

Monoamine oxidase A (MAOA) is an enzyme expressed in the brain that metabolizes dopamine, norepinephrine, epinephrine, and serotonin. Abnormalities of serotonin neurotransmission have long been implicated in the psychopathology of autism. A polymorphism exists within the promoter region of the MAOA gene that influences MAOA expression levels so that “low activity” alleles are associated with increased neurotransmitter levels in the brain. Individuals with autism often exhibit elevated serotonin levels. Additional studies indicate that the “low activity” allele may be associated with lower IQ and more severe autistic symptoms. In this study we genotyped the MAOA promoter polymorphism in a group of 29 males (age 2–3 years) with autism and a group of 39 healthy pediatric controls for whom brain MRI data was available. We found a consistent association between the “low activity” allele and larger brain volumes for regions of the cortex in children with autism but not in controls. We did not find evidence for over-transmission of the “low activity” allele in a separate sample of 114 affected sib pairfamilies. Nor did we find any unknown SNPs in yet another sample of 96 probands. Future studies will determine if there is a more severe clinical phenotype associated with both the “low activity” genotype and the larger brain volumes in our sample

Posterior probability of linkage analysis of autism dataset identifies linkage to chromosome 16

To apply phenotypic and statistical methods designed to account for heterogeneity to linkage analyses of the autism Collaborative Linkage Study of Autism (CLSA) affected sibling pair families

Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma

PURPOSE. Mutations in the myocilin gene (MYOC) are the most common known genetic cause of primary open-angle glaucoma (POAG). The purpose of this study was to determine whether topical ocular sodium 4-phenylbutyrate (PBA) treatment rescues glaucoma phenotypes in a mouse model of myocilin-associated glaucoma (Tg-MYOC Y437H mice). METHODS. Tg-MYOC Y437H mice were treated with PBA eye drops (n ϭ 10) or sterile PBS (n ϭ 8) twice daily for 5 months. Long-term safety and effectiveness of topical PBA (0.2%) on glaucoma phenotypes were examined by measuring intraocular pressure (IOP) and pattern ERG (PERG), performing slit lamp evaluation of the anterior chamber, analyzing histologic sections of the anterior segment, and comparing myocilin levels in the aqueous humor and trabecular meshwork of Tg-MYOC Y437H mice. Sci. 2012;53: 1557-1565 RESULTS. Tg-MYO

A Peripherin/Retinal Degeneration Slow Mutation (Pro-210-Arg) Associated with Macular and Peripheral Retinal Degeneration

BackgroundMutations in the peripherin/retinal degeneration slow (RDS) gene have been identified in patients with retinitis pigmentosa and pattern macular dystrophy. The authors initially examined a large family affected with both peripheral and macular degeneration, inherited as an autosomal dominant trait. Screening for peripherin/RDS mutations identified a previously unreported nucleotide alteration in all of the affected individuals. Two additional families later were found to have this same mutation.MethodsDNA samples from the members of three unrelated families were screened for peripherin/RDS mutations by denaturing gradient gel electrophoresis of the polymerase chain reaction-amplified peripherin/RDS coding sequences. The sequence change that was detected was further characterized by DNA sequencing. Family members were examined and evaluated with psychophysical and electrophysiologic methods.ResultsA proline to arginine mutation in codon 210 of peripherin/RDS was found in all clinically affected individuals. Macular changes included extensive geographic atrophy, pigment epithelial changes, and/or drusen. The proline to arginine mutation was not found among 100 healthy individuals, making it unlikely to be a nondisease-causing polymorphism.ConclusionsThe authors identified a novel peripherin/RDS gene mutation associated with autosomal dominant retinal degeneration in patients from three different families. The largest family showed a broad variability in the expressivity of the mutation. The overlap of clinical features with those of age-related maculopathy highlights the need to consider photoreceptor-specific genes as potential factors in the etiology of the latter condition

Deducing the pathogenic contribution of recessive ABCA4 alleles in an outbred population

Accurate prediction of the pathogenic effects of specific genotypes is important for the design and execution of clinical trials as well as for meaningful counseling of individual patients. However, for many autosomal recessive diseases, it can be difficult to deduce the relative pathogenic contribution of individual alleles because relatively few affected individuals share the same two disease-causing variations. In this study, we used multiple regression analysis to estimate the pathogenicity of specific alleles of ABCA4 in patients with retinal phenotypes ranging from Stargardt disease to retinitis pigmentosa. This analysis revealed quantitative allelic effects on two aspects of the visual phenotype, visual acuity (P < 10−3) and visual field (P < 10−7). Discordance between visual acuity and visual field in individual patients suggests the existence of at least two non-ABCA4 modifying factors. The findings of this study will facilitate the discovery of factors that modify ABCA4 disease and will also aid in the optimal selection of subjects for clinical trials of new therapies

Osteoarthritis-Like Changes in Bardet–Biedl Syndrome Mutant Ciliopathy Mice (Bbs1M390R/M390R): Evidence for a Role of Primary Cilia in Cartilage Homeostasis and Regulation of Inflammation

Osteoarthritis (OA) is a debilitating inflammation related disease characterized by joint pain and effusion, loss of mobility, and deformity that may result in functional joint failure and significant impact on quality of life. Once thought of as a simple “wear and tear” disease, it is now widely recognized that OA has a considerable metabolic component and is related to chronic inflammation. Defects associated with primary cilia have been shown to be cause OA-like changes in Bardet–Biedl mice. We examined the role of dysfunctional primary cilia in OA in mice through the regulation of the previously identified degradative and pro-inflammatory molecular pathways common to OA. We observed an increase in the presence of pro-inflammatory markers TGFβ-1 and HTRA1 as well as cartilage destructive protease MMP-13 but a decrease in DDR-2. We observed a morphological difference in cartilage thickness in Bbs1M390R/M390R mice compared to wild type (WT). We did not observe any difference in OARSI or Mankin scores between WT and Bbs1M390R/M390R mice. Primary cilia appear to be involved in the upregulation of biomarkers, including pro-inflammatory markers common to OA

Requirement of Bardet-Biedl syndrome proteins for leptin receptor signaling

Obesity is a major public health problem in most developed countries and a major risk factor for diabetes and cardiovascular disease. Emerging evidence indicates that ciliary dysfunction can contribute to human obesity but the underlying molecular and cellular mechanisms are unknown. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous human obesity syndrome associated with ciliary dysfunction. BBS proteins are thought to play a role in cilia function and intracellular protein/vesicle trafficking. Here, we show that BBS proteins are required for leptin receptor (LepR) signaling in the hypothalamus. We found that Bbs2−/−, Bbs4−/− and Bbs6−/− mice are resistant to the action of leptin to reduce body weight and food intake regardless of serum leptin levels and obesity. In addition, activation of hypothalamic STAT3 by leptin is significantly decreased in Bbs2−/−, Bbs4−/− and Bbs6−/− mice. In contrast, downstream melanocortin receptor signaling is unaffected, indicating that LepR signaling is specifically impaired in Bbs2−/−, Bbs4−/− and Bbs6−/− mice. Impaired LepR signaling in BBS mice was associated with decreased Pomc gene expression. Furthermore, we found that BBS1 protein physically interacts with the LepR and that loss of BBS proteins perturbs LepR trafficking. Our data indicate that BBS proteins mediate LepR trafficking and that impaired LepR signaling underlies energy imbalance in BBS. These findings represent a novel mechanism for leptin resistance and obesity