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Proteasome 26S, the eukaryotic proteasome, serves as the machinery for cellular protein degradation. It is composed of the 20S core particle and one or two 19S regulatory particles, composed of a base and a lid. To date, several human diseases have been associated with mutations within the 26S proteasome subunits; only one of them affects a base subunit. We now delineate an autosomal recessive syndrome of failure to thrive, severe developmental delay and intellectual disability, spastic tetraplegia with central hypotonia, chorea, hearing loss, micropenis and undescended testes, as well as mild elevation of liver enzymes. None of the affected individuals achieved verbal communication or ambulation. Ventriculomegaly was evident on MRI. Homozygosity mapping combined with exome sequencing revealed a disease-associated p.I328T PSMC1 variant. Protein modeling demonstrated that the PSMC1 variant is located at the highly conserved putative ATP binding and hydrolysis domain, and is suggested to interrupt a hydrophobic core within the protein. Fruit flies in which we silenced the Drosophila ortholog Rpt2 specifically in the eye exhibited an apparent phenotype that was highly rescued by the human wild-type PSMC1, yet only partly by the mutant PSMC1, proving the functional effect of the p.I328T disease-causing variant.

Congenital myopathies are a broad group of inborn muscle disorders caused by a multitude of genetic factors, often characterized by muscle atrophy and hypotonia.Clinical studies, imaging, histology, whole-exome sequencing (WES) and muscle tissue RNA studies.We describe a severe congenital myopathy manifesting at birth with bilateral clubfeet, delayed motor development and hypotonia, becoming evident by 4 months of age. At 3 years of age, the patient had tongue fasciculations, was bedridden, and was chronically ventilated via tracheostomy. Imaging studies demonstrated severe muscle atrophy and, surprisingly, cerebral atrophy; electromyography demonstrated a myasthenic pattern and histological evaluation did not facilitate a definitive diagnosis. Trio WES did not identify a causative variant, except for a non-canonical intronic TPM3 c.118-12GA variant of uncertain significance. Transcript analysis of muscle tissue from the patient proved the pathogenicity of this homozygous variant, with a 97% reduction in the muscle-specific TPM3.12 transcript.This study broadens the phenotypic spectrum of recessive TPM3 disease, highlighting tongue fasciculations and bilateral clubfoot, as well as possibly-related cerebral atrophy. It also shows the importance of a broad approach to genetic analysis and the utility of RNA-based studies, demonstrating efficacy of early genome and transcriptome queries in facilitating rapid and cost-effective diagnosis of congenital myopathies.

To assess the feasibility of Fractional exhaled Nitric Oxide (FeNO) as a simple, non-invasive, cost-effective and portable biomarker and decision support tool for risk stratification of COVID-19 patients.We conducted a single-center prospective cohort study of COVID-19 patients whose FeNO levels were measured upon ward admission by the Vivatmo-me handheld device. Demographics, COVID-19 symptoms, and relevant hospitalization details were retrieved from the hospital databases. The patients were divided into those discharged to recover at home and those who died during hospitalization or required admission to an intensive care unit, internal medicine ward, or dedicated facility (severe outcomes group).Fifty-six patients were enrolled. The only significant demographic difference between the severe outcomes patients (n = 14) and the home discharge patients (n = 42) was age (64.21 ± 13.97 vs. 53.98 ± 15.57 years, respectively, P = .04). The admission FeNO measurement was significantly lower in the former group compared with the latter group (15.86 ± 14.74 vs. 25.77 ± 13.79, parts per billion [PPB], respectively, P = .008). Time to severe outcome among patients with FeNO measurements ≤11.8 PPB was significantly shorter compared with patients whose FeNO measured11.8 PPB (19.25 ± 2.96 vs. 24.41 ± 1.09 days, respectively, 95% confidence interval [CI] 1.06 to 4.25). An admission FeNO ≤11.8 PPB was a significant risk factor for severe outcomes (odds ratio = 12.8, 95% CI: 2.78 to 58.88, P = .001), with a receiver operating characteristics curve of 0.752.FeNO measurements by the Vivatmo-me handheld device can serve as a biomarker and COVID-19 support tool for medical teams. These easy-to-use, portable, and noninvasive devices may serve as valuable ED bedside tools during a pandemic.

Six individuals of consanguineous Bedouin kindred presented at infancy with an autosomal recessive syndrome of severe global developmental delay, positive pyramidal signs, unique dysmorphism, skeletal abnormalities, and severe failure to thrive with normal birth weights. Patients had a profound intellectual disability and cognitive impairment with almost no acquired developmental milestones by 12 months. Early-onset axial hypotonia evolved with progressive muscle weakness, reduced muscle tone, and hyporeflexia. Craniofacial dysmorphism consisted of a triangular face with a prominent forehead and midface hypoplasia. Magnetic resonance imaging (MRI) demonstrated thinning of the corpus callosum and paucity of white matter. Genome-wide linkage analysis identified a single ~4 Mbp disease-associated locus on chromosome 7q21.13-q21.3 (LOD score5). Whole-exome and genome sequencing identified no nonsynonymous pathogenic biallelic variants in any of the genes within this locus. Following the exclusion of partially resembling syndromes, we now describe a novel autosomal recessive syndrome mapped to a ~4Mbp locus on chromosome 7.

Primary microcephaly and Seckel syndrome are rare genetically and clinically heterogenous brain development disorders. Several exonic/splicing mutations are reported for these disorders to date, but ∼40% of all cases remain unexplained. We aimed to uncover the genetic correlate(s) in a family of multiple siblings with microcephaly. A novel homozygous intronic variant (NC_000013.10:g.25459823TC) in CENPJ (13q12) segregating with all four affected male siblings was identified by exome sequencing and validated by targeted linkage approach (logarithm of the odds score 1.8 at θ 0.0). RT-PCR of CENPJ in affected siblings using their EBV derived cell lines showed aberrant transcripts suggestive of exon skipping confirmed by Sanger sequencing. Significantly reduced wild type transcript/protein in the affected siblings having the splice variant indicates a leaky gene expression of pathological relevance. Based on known CENPJ function, assessing for mitotic alterations revealed defect in centrosome duplication causing mono/multicentrosome(s) at prophase, delayed metaphase, and unequal chromosomal segregation in patient cells. Clinical features witnessed in this study expand the spectrum of CENPJ-associated primary microcephaly and Seckel syndrome. Furthermore, besides the importance of regulatory variants in classical monogenic disorders these findings provide new insights into splice site biology with possible implications for ASO-based therapies.

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.

Mutations in myotubularin-related protein 2 (MTMR2) were shown to underlie Charcot-Marie-Tooth type 4B1 (CMT4B1) disease, a rare autosomal recessive demyelinating neuropathy, characterized by severe early-onset motor and sensory neuropathy. We describe three siblings of consanguineous kindred presenting with hypotonia, reduced muscle tone, action tremor, dysmetria, areflexia, and skeletal deformities, consistent with a diagnosis of CMT. Whole-exome sequencing identified a novel homozygous c.336_337 insertion mutation in MTMR2, resulting in a frameshift and putative truncated protein. In this concise report, we discuss the clinical presentation of this rare disease and support the limited number of observations regarding the pathogenesis of MTMR2-related neuropathies.

COX15 mutations were shown to underlie Leigh syndrome (LS), a progressive subacute necrotizing encephalopathy caused by defects in the mitochondrial respiratory chain. Here, two siblings of consanguineous kindred presented in infancy with a syndrome of hypotonia, nystagmus, psychomotor retardation, and pyramidal signs. Toward the end of their second year, both patients developed progressive quadriparesis, convulsions, and pseudobulbar palsy. Similar to two previously reported cases, one of the two affected siblings had severe hypertrophic obstructive cardiomyopathy, hearing loss, and no visual response. Through linkage analysis and whole-exome sequencing, we identified a homozygous p.R217W mutation in Cytochrome C oxidase assembly protein COX15 homolog. Consistent with the known heterogeneity of mitochondrial diseases in general and that of LS in particular, several phenotypic features were markedly distinguished between the affected siblings and in relation to previous reports of COX15 mutations. Interestingly, of the previously reported five cases of COX15-mutated patients, all of different ethnic origins, three had a p.R217W mutation. We highlight p.R217W as a hotspot mutation in COX15 and delineate the phenotypic variability, both between the patients we describe and in all cases reported to date.

Gout is caused by deposition of monosodium urate crystals in joints when plasma uric acid levels are chronically elevated beyond the saturation threshold, mostly due to renal underexcretion of uric acid. Although molecular pathways of this underexcretion have been elucidated, its etiology remains mostly unknown. We demonstrate that gout can be caused by a mutation in LDHD within the putative catalytic site of the encoded d-lactate dehydrogenase, resulting in augmented blood levels of d-lactate, a stereoisomer of l-lactate, which is normally present in human blood in miniscule amounts. Consequent excessive renal secretion of d-lactate in exchange for uric acid reabsorption culminated in hyperuricemia and gout. We showed that LDHD expression is enriched in tissues with a high metabolic rate and abundant mitochondria and that d-lactate dehydrogenase resides in the mitochondria of cells overexpressing the human LDHD gene. Notably, the p.R370W mutation had no effect on protein localization. In line with the human phenotype, injection of d-lactate into naive mice resulted in hyperuricemia. Thus, hyperuricemia and gout can result from the accumulation of metabolites whose renal excretion is coupled to uric acid reabsorption.

Four affected individuals of consanguineous kindred presented at infancy with an apparently autosomal recessive syndrome of polyuria and hypokalemic metabolic alkalosis, following maternal polyhydramnios and premature delivery, culminating in severe failure to thrive. Hypercalciuria, nephrocalcinosis, and hyperaldosteronism were further apparent as well as an unusual finding of intermittent hypernatremia. Additionally, all patients demonstrated variable micrognathia with upper respiratory airway abnormalities. As neither postnatal hyperkalemia nor permanent hearing deficits were shown, clinical assessment was consistent with antenatal Bartter syndrome (ABS) type I, which was never described before in the Israeli Bedouin population. Through genome-wide linkage analysis, we identified a single ∼3.3 Mbp disease-associated locus on chromosome 15q21.1, segregating within the pedigree. Whole-exome sequencing revealed a single novel homozygous missense mutation within this locus, in SLC12A1, encoding the Na-K-Cl cotransporter, NKCC2, in accordance with the clinical diagnosis. In this concise study, we report a novel missense mutation within the SLC12A1 gene, causing a severe form of ABS type I, the first to be described in Israeli Bedouins, with unusual clinical features of hypernatremia caused by nephrogenic diabetes insipidus and putatively related micrognathia with upper airway abnormalities .

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.

Genetic studies of Mendelian and rare diseases face the critical challenges of identifying pathogenic gene variants and their modes-of-action. Previous efforts rarely utilized the tissue-selective manifestation of these diseases for their elucidation. Here we introduce an interpretable machine learning (ML) platform that utilizes heterogeneous and large-scale tissue-aware datasets of human genes, and rigorously, concurrently and quantitatively assesses hundreds of candidate mechanisms per disease. The resulting tissue-aware ML platform is applicable in gene-specific, tissue-specific, or patient-specific modes. Application of the platform to selected Mendelian disease genes pinpointed mechanisms that lead to tissue-specific disease manifestation. When applied jointly to diseases that manifest in the same tissue, the models revealed common known and previously underappreciated factors that underlie tissue-selective disease manifestation. Lastly, we harnessed our ML platform toward genetic diagnosis of tissue-selective rare diseases. Patient-specific models of candidate disease-causing genes from 50 patients successfully prioritized the pathogenic gene in 86% of the cases, implying that the tissue-selectivity of rare diseases aids in filtering out unlikely candidate genes. Thus, interpretable tissue-aware ML models can boost mechanistic understanding and genetic diagnosis of tissue-selective heritable diseases. A webserver supporting gene prioritization is available at https://netbio.bgu.ac.il/trace/.

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.

Sphingolipidoses are monogenic lipid storage diseases caused by variants in enzymes of lipid synthesis and metabolism. We describe an autosomal recessive complex neurological disorder affecting consanguineous kindred. All four affected individuals, born at term following normal pregnancies, had mild to severe intellectual disability, spastic quadriplegia, scoliosis and epilepsy in most, with no dysmorphic features. Brain MRI findings were suggestive of leukodystrophy, with abnormal hyperintense signal in the periventricular perioccipital region and thinning of the body of corpus callosum. Notably, all affected individuals were asymptomatic at early infancy and developed normally until the age of 8–18 months, when deterioration ensued. Homozygosity mapping identified a single 8.7 Mb disease-associated locus on chromosome 1q41–1q42.13 between rs1511695 and rs537250 (two-point LOD score 2.1). Whole exome sequencing, validated through Sanger sequencing, identified within this locus a single disease-associated homozygous variant in DEGS1, encoding C4-dihydroceramide desaturase, an enzyme of the ceramide synthesis pathway. The missense variant, segregating within the family as expected for recessive heredity, affects an evolutionary-conserved amino acid of all isoforms of DEGS1 (c.656A>G, c.764A>G; p.(N219S), p.(N255S)) and was not found in a homozygous state in ExAC and gnomAD databases or in 300 ethnically matched individuals. Lipidomcs analysis of whole blood of affected individuals demonstrated augmented levels of dihydroceramides, dihydrosphingosine, dihydrosphingosine-1-phosphate and dihydrosphingomyelins with reduced levels of ceramide, sphingosine, sphingosine-1-phosphate and monohexosylceramides, as expected in malfunction of C4-dihydroceramide desaturase. Thus, we describe a sphingolipidosis causing a severe regressive neurological disease.

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.

Autologous blood-derived products (ABP) are the focus of growing scientific interest and are investigated and used for multiple medical indications. ABPs hold promise thanks to their availability, ease of preparation, and low risk of adverse allogenic reaction, hypersensitivity, and contamination. Compositional analysis of ABPs reveals a diverse mixture of cellular components, cytokines and growth factors that play roles in healing processes such as tissue proliferation and angiogenesis, modulation of the local environment through chemotaxis and regulation of inflammation and the extracellular matrix, as well as several immunomodulatory actions. Thus, the administration of ABP induces supraphysiological levels of components necessary for orchestrating reparative efforts in currently difficult-to-treat medical conditions. In this article, we review the variety of autologous blood-derived products, their composition, current clinical uses, regulatory climate, and mechanisms of action.

Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.

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.