Studies of ciliopathies have served in elucidating much of our knowledge of structure and function of primary cilia. We report humans with Bardet-Biedl syndrome who display intellectual disability, retinitis pigmentosa, obesity, short stature and brachydactyly, stemming from a homozyogous truncation mutation in SCAPER, a gene previously associated with mitotic progression. Our findings, based on linkage analysis and exome sequencing studies of two remotely related large consanguineous families, are in line with recent reports of SCAPER variants associated with intellectual disability and retinitis pigmentosa. Using immuno-fluorescence and live cell imaging in NIH/3T3 fibroblasts and SH-SY5Y neuroblastoma cell lines over-expressing SCAPER, we demonstrate that both wild type and mutant SCAPER are expressed in primary cilia and co-localize with tubulin, forming bundles of microtubules. While wild type SCAPER was rarely localized along the ciliary axoneme and basal body, the aberrant protein remained sequestered to the cilia, mostly at the ciliary tip. Notably, longer cilia were demonstrated both in human affected fibroblasts compared to controls, as well as in NIH/3T3 cells transfected with mutant versus wildtype SCAPER. As SCAPER expression is known to peak at late G1 and S phase, overlapping the timing of ciliary resorption, our data suggest a possible role of SCAPER in ciliary dynamics and disassembly, also affecting microtubule-related mitotic progression. Thus, we outline a human ciliopathy syndrome and demonstrate that it is caused by a mutation in SCAPER, affecting primary cilia.

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Twelve individuals of consanguineous Bedouin kindred presented with autosomal recessive progressive spastic paraplegia evident as of age 0-24 months, with spasticity of lower limbs, hyperreflexia, toe walking and equinus deformity. Kyphoscolisois was evident in older patients. Most had atrophy of the lateral aspects of the tongue and few had intellectual disability. Nerve conduction velocity, electromyography and head and spinal cord magnetic resonance imaging were normal in tested subjects. Muscle biopsy showed occasional central nuclei and fiber size variability with small angular fibers. Genome-wide linkage analysis identified a 6.7Mbp disease-associated locus on chromosome 3q21.3-3q22.2 (LOD score 9.02; D3S1290). Whole-exome sequencing identified a single homozygous variant within this locus, c.51_52ins(28); p.(V18fs56*) in KY, segregating in the family as expected and not found in 190 Bedouin controls. High KY transcript levels were demonstrated in muscular organs with lower expression in the CNS. The phenotype is reminiscent of kyphoscoliosis seen in Ky null mice. Two recent studies done independently and parallel to ours describe somewhat similar phenotypes in one and two patients with KY mutations. KY encodes a tranglutaminase-like peptidase, which interacts with muscle cytoskeletal proteins and is part of a Z-band protein complex, suggesting the disease mechanism may resemble myofibrillar myopathy. However, the mixed myopathic-neurologic features caused by human and mouse Ky mutations are difficult to explain by loss of KY sarcomere stabilizing function alone. KY transcription in CNS tissues may imply that it also has a role in neuromotor function, in line with the irregularity of neuromuscular junction in Ky null mutant mice.

Background: Paroxysmal atrial fibrillation (AF) can be caused by gain-of-function mutations in genes, encoding the cardiac potassium channel subunits KCNJ2 , KCNE1 , and KCNH2 that mediate the repolarizing potassium currents I k1 , I ks , and I kr , respectively. Methods: Linkage analysis, whole-exome sequencing, and Xenopus oocyte electrophysiology studies were used in this study. Results: Through genetic studies, we showed that autosomal dominant early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in KCND2 , encoding the pore-forming (α) subunit of the Kv4.2 cardiac potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac fast transient outward K + current, I to . I to underlies the early phase of repolarization in the cardiac action potential, thereby setting the initial potential of the plateau phase and governing its duration and amplitude. In Xenopus oocytes, the mutation increased the channel’s inactivation time constant and affected its regulation: p.S447 resides in a protein kinase C (PKC) phosphorylation site, which normally allows attenuation of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired response to PKC; hence, Kv4.2 membrane expression was augmented, enhancing potassium currents. Coexpression of mutant and wild-type channels (recapitulating heterozygosity in affected individuals) showed results similar to the mutant channel alone. Finally, in a hybrid channel composed of Kv4.3 and Kv4.2, simulating the mature endogenous heterotetrameric channel underlying I to , the p.S447R Kv4.2 mutation exerted a gain-of-function effect on Kv4.3. Conclusions: The mutation alters Kv4.2’s kinetic properties, impairs its inhibitory regulation, and exerts gain-of-function effect on both Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects presumably increase the repolarizing potassium current I to , thereby abbreviating action potential duration, creating arrhythmogenic substrate for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to demonstrate circadian variation, with peak expression at daytime in murine hearts (human nighttime), with possible relevance to the nocturnal onset of paroxysmal AF symptoms in our patients. The atrial-specific phenotype suggests that targeting Kv4.2 might be effective in the treatment of nocturnal paroxysmal AF, avoiding adverse ventricular effects.

BackgroundConsanguineous kindred presented with an autosomal recessive syndrome of intrauterine growth retardation, marked developmental delay, spastic quadriplegia with profound contractures, pseudobulbar palsy with recurrent aspirations, epilepsy, dysmorphism, neurosensory deafness and optic nerve atrophy with no eye fixation. Affected individuals died by the age of 4. Brain MRI demonstrated microcephaly, semilobar holoprosencephaly and agenesis of corpus callosum. We aimed at elucidating the molecular basis of this disease.MethodsGenome-wide linkage analysis combined with whole exome sequencing were performed to identify disease-causing variants. Functional consequences were investigated in fruit flies null mutant for the Drosophila SEC31A orthologue. SEC31A knockout SH-SY5Y and HEK293T cell-lines were generated using CRISPR/Cas9 and studied through qRT-PCR, immunoblotting and viability assays.ResultsThrough genetic studies, we identified a disease-associated homozygous nonsense mutation in SEC31A. We demonstrate that SEC31A is ubiquitously expressed, and that the mutation triggers nonsense-mediated decay of its transcript, comprising a practical null mutation. Similar to the human disease phenotype, knockdown SEC31A flies had defective brains and early lethality. Moreover, in line with SEC31A encoding one of the two coating layers comprising the Coat protein complex II (COP-II) complex, trafficking newly synthesised proteins from the endoplasmic reticulum (ER) to the Golgi, CRISPR/Cas9-mediated SEC31A null mutant cells demonstrated reduced viability through upregulation of ER-stress pathways.ConclusionWe demonstrate through human and Drosophila genetic and in vitro molecular studies, that a severe neurological syndrome is caused by a null mutation in SEC31A, reducing cell viability through enhanced ER-stress response, in line with SEC31A’s role in the COP-II complex.

BackgroundThe hypothalamic G-protein-coupled-receptor melanocortin-4 receptor (MC4R) is a key player in the central circuit regulating energy expenditure and appetite. Heterozygous loss-of-function MC4R mutations are the most common known genetic cause of monogenic human obesity, with more than 200 mutations described to date, affecting 2–3% of the population in various cohorts tested. Homozygous or compound heterozygous MC4R mutations are much less frequent, and only few families have been described in which heterozygotes and homozygotes of the same mutation are found.MethodsWe performed exome sequencing in a consanguineous Bedouin family with morbid obesity to identify the genetic cause of the disease. Clinical examination and biochemical assays were done to delineate the phenotype.ResultsWe report the frequency of MC4R mutations in the large inbred Bedouin Israeli population. Furthermore, we describe consanguineous inbred Bedouin kindred with multiple individuals that are either homozygous or heterozygous carries of the same novel MC4R mutation (c.124G > T, p.E42*). All family members with the homozygous mutation exhibited morbid early-onset obesity, while heterozygote individuals had either a milder overweight phenotype or no discernable phenotype compared to wild type family members. While elder individuals homozygous or heterozygous for the MC4R mutation had abnormally high triglycerides, cholesterol, glucose and HbA1C levels, most did not.ConclusionsMC4R mutation homozygotes exhibited morbid early-onset obesity, while heterozygotes had a significantly milder overweight phenotype. Whereas obesity due to MC4R mutations is evident as of early age – most notably in homozygotes, the metabolic consequences emerge only later in life.

Attention-deficit hyperactivity disorder (ADHD) is a common childhood-onset psychiatric disorder characterized by inattention, impulsivity and hyperactivity. ADHD exhibits substantial heritability, with rare monogenic variants contributing to its pathogenesis. Here we demonstrate familial ADHD caused by a missense mutation in CDH2, which encodes the adhesion protein N-cadherin, known to play a significant role in synaptogenesis; the mutation affects maturation of the protein. In line with the human phenotype, CRISPR/Cas9-mutated knock-in mice harboring the human mutation in the mouse ortholog recapitulated core behavioral features of hyperactivity. Symptoms were modified by methylphenidate, the most commonly prescribed therapeutic for ADHD. The mutated mice exhibited impaired presynaptic vesicle clustering, attenuated evoked transmitter release and decreased spontaneous release. Specific downstream molecular pathways were affected in both the ventral midbrain and prefrontal cortex, with reduced tyrosine hydroxylase expression and dopamine levels. We thus delineate roles for CDH2-related pathways in the pathophysiology of ADHD.

Myopathy is the main adverse effect of the widely prescribed statin drug class. Statins exert their beneficial effect by inhibiting HMG CoA-reductase, the rate-controlling enzyme of the mevalonate pathway. The mechanism of statin myopathy is yet to be resolved, and its treatment is insufficient. Through homozygosity mapping and whole exome sequencing, followed by functional analysis using confocal microscopy and biochemical and biophysical methods, we demonstrate that a distinct form of human limb girdle muscular disease is caused by a pathogenic homozygous loss-of-function missense mutation in HMG CoA reductase ( HMGCR ), encoding HMG CoA-reductase . We biochemically synthesized and purified mevalonolactone, never administered to human patients before, and establish the safety of its oral administration in mice. We then show that its oral administration is effective in treating a human patient with no significant adverse effects. Furthermore, we demonstrate that oral mevalonolactone resolved statin-induced myopathy in mice. We conclude that HMGCR mutation causes a late-onset severe progressive muscular disease, which shows similar features to statin-induced myopathy. Our findings indicate that mevalonolactone is effective both in the treatment of hereditary HMGCR myopathy and in a murine model of statin myopathy. Further large clinical trials are in place to enable the clinical use of mevalonolactone both in the rare orphan disease and in the more common statin myopathy.