OffRisk: a docker image for annotating CRISPR off-target sites in the human genome.

Summary: The CRISPR-Cas9 system has been adapted to achieve targeted genome editing as well as transcriptional control by customizing 20-nt guide RNA (gRNA) molecules for desired regions in the target genome. Designing gRNAs must consider nonspecific and unintended binding, known as off-targets, since these may have potentially harmful effects. To assist in gRNA design, we have developed OffRisk. This Docker-based tool annotates off-target sites in the human genome and assigns them a potential risk label by incorporating functional and regulatory information at different molecular levels. Availability and implementation: OffRisk is available at https://github.com/IsanaVekslerLublinsky/OffRisk and https://github.com/IsanaVekslerLublinsky/OffRisk-ui (including code, user guide, docker installation guide, and running examples).All processed datasets are available at https://zenodo.org/record/8289271.

Correction to: ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis.

After the publication of the original article [1], we were notified the upper panel of the Fig. 1, where the patients' codes are listed, was cropped by mistake so the patients 1-8 are repeated.

Whole exome sequencing (WES) approach for diagnosing primary immunodeficiencies (PIDs) in a highly consanguineous community.

Primary immunodeficiencies (PIDs) are a heterogeneous group of monogenic inborn errors of immunity. The genetic causes of these diseases can be identified using whole exome sequencing (WES). Here, DNA samples from 106 patients with a clinical suspicion of PID were subjected to WES in order to test the diagnostic yield of this test in a highly consanguineous community. A likely genetic diagnosis was achieved in 70% of patients. Several factors were considered to possibly influence the diagnostic rate of WES among our cohort including early age, presence of consanguinity, family history suggestive of PID, the number of family members who underwent WES and the clinical phenotype of the patient. The highest diagnostic rate was in patients with combined immunodeficiency or with a syndrome. Notably, WES findings altered the clinical management in 39% (41/106) of patients in our cohort. Our findings support the use of WES as an important diagnostic tool in patients with suspected PID, especially in highly consanguineous communities.

ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis.

BACKGROUND: Emerging mutations in the ESR1 gene that encodes for the estrogen receptor (ER) are associated with resistance to endocrine therapy. ESR1 mutations rarely exist in primary tumors (~ 1%) but are relatively common (10-50%) in metastatic, endocrine therapy-resistant cancers and are associated with a shorter progression-free survival. Little is known about the incidence and clinical implication of these mutations in early recurrence events, such as local recurrences or newly diagnosed metastatic disease. METHODS: We collected 130 archival tumor samples from 103 breast cancer patients treated with endocrine therapy prior to their local/metastatic recurrence. The cohort consisted of 41 patients having at least 1 sample from local/loco-regional recurrence and 62 patients with metastatic disease (of whom 41 newly diagnosed and 28 with advanced disease). The 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) were analyzed either by targeted sequencing or by droplet digital PCR. Progression-free survival (PFS), disease-free survival (DFS), and distant recurrence-free survival (DRFS) were statistically tested by Kaplan-Meier analysis. RESULTS: The prevalence of ESR1 mutations was 5/41 (12%) in newly diagnosed metastatic patients and 5/28 (18%) for advanced metastases, detected at allele frequency > 1%. All mutations in advanced metastases were detected in patients previously treated with both tamoxifen (TAM) and aromatase inhibitors (AI). However, in newly diagnosed metastatic patients, 4/5 mutations occurred in patients treated with TAM alone. PFS on AI treatment in metastatic patients was significantly shorter for ESR1 mutation carriers (p = 0.017). In the local recurrence cohort, ESR1 mutations were identified in 15/41 (36%) patients but only 4/41 (10%) were detected at allele frequency > 1%. Again, most mutations (3/4) were detected under TAM monotherapy. Notably, 1 patient developed ESR1 mutation while on neoadjuvant endocrine therapy. DFS and DRFS were significantly shorter (p = 0.04 and p = 0.017, respectively) in patients that had ESR1 mutations (> 1%) in their loco-regional recurrence tumor. CONCLUSIONS: Clinically relevant ESR1 mutations are prevalent in newly diagnosed metastatic and local recurrence of endocrine-treated breast cancer. Since local recurrences are amenable to curative therapy, these mutations may inform the selection of subsequent endocrine therapies.

Reduced Function and Diversity of T Cell Repertoire and Distinct Clinical Course in Patients With IL7RA Mutation.

The alpha subunit of IL-7 receptor (IL7R7α) is critical for the differentiation of T cells, specifically for the development and maintenance of γδT cells. Mutations in IL7RA are associated with Severe Combined Immunodeficiency (SCID). Infants with IL7RA deficiency can be identified through newborn screening program. We aimed at defining the immunological and genetic parameters that are directly affected by the IL7RA mutation on the immune system of five unrelated patients which were identified by our newborn screening program for SCID. The patients were found to have a novel identical homozygote mutation in IL7RA (n.c.120 C>G; p.F40L). Both surface expression of IL7Rα and functionality of IL-7 signaling were impaired in patients compared to controls. Structural modeling demonstrated instability of the protein structure due to the mutation. Lastly the TRG immune repertoire of the patients showed reduced diversity, increased clonality and differential CDR3 characteristics. Interestingly, the patients displayed significant different clinical outcome with two displaying severe clinical picture of immunodeficiency and three had spontaneous recovery. Our data supports that the presented IL7RA mutation affects the IL-7 signaling and shaping of the TRG repertoire, reinforcing the role of IL7RA in the immune system, while non-genetic factors may exist that attribute to the ultimate clinical presentation and disease progression.

c.259A>C in the fibrinogen gene of alpha chain (FGA) is a fibrinogen with thrombotic phenotype.

INTRODUCTION: Dysfibrinogenemia is a rare inherited disease that results from mutation in one of the three fibrinogen genes. Diagnosis can be misleading since it may present as a bleeding tendency or thrombosis and a specific coagulation test for diagnosis is not routinely available. AIM: To search for a new candidate gene of thrombophilia in a family with three generations of arterial and venous thrombosis. METHODS: Whole exome sequencing followed by Sanger validation and segregation analysis was carried out. In addition, structural modeling was performed. Screening for thrombophilia along with blood counts, prothrombin time, activated partial thromboplastin, thrombin, reptilase time, and fibrinogen was done in each patient. RESULTS AND DISCUSSION: A missense c.259A>C, p.K87Q (g.chr4: 155510050A-C) (rs764281241) in FGA gene was found in all three siblings without any other known thrombophilia marker to explain thrombosis in all three siblings. It is expected to be damaging by six out of seven prediction programs and is very rare in the entire population with Exac=0.000008. CONCLUSION: The occurrence of the c.259A>C mutation in FGA may well explain the thrombosis phenotype of the affected family and is suggested as a new marker for thrombophilia phenotype.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

Somatic NRAS mutation in patient with generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA or lymphangiomatosis) is a rare disease characterized by a diffuse proliferation of lymphatic vessels in skin and internal organs. It often leads to progressive respiratory failure and death, but its etiology is unknown. Here, we isolated lymphangiomatosis endothelial cells from GLA tissue. These cells were characterized by high proliferation and survival rates, but displayed impaired capacities for migration and tube formation. We employed whole exome sequencing to search for disease-causing genes and identified a somatic mutation in NRAS. We used mouse and zebrafish model systems to initially evaluate the role of this mutation in the development of the lymphatic system, and we studied the effect of drugs blocking the downstream effectors, mTOR and ERK, on this disease.

Whole-genome sequencing reveals principles of brain retrotransposition in neurodevelopmental disorders.

Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are "non-classical" in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as "lightning rods" for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.

RNA editing by ADAR1 leads to context-dependent transcriptome-wide changes in RNA secondary structure.

Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.

Congenital Sucrase-isomaltase Deficiency: A Novel Compound Heterozygous Mutation Causing Aberrant Protein Localization.

OBJECTIVES: Congenital diarrheal disorders is a group of inherited enteropathies presenting in early life and requiring parenteral nutrition. In most cases, genetics may be the key for precise diagnosis. We present an infant girl with chronic congenital diarrhea that resolved after introduction of fructose-based formula but had no identified mutation in the SLC5A1 gene. Using whole exome sequencing (WES) we identified other mutations that better dictated dietary adjustments. METHODS: WES of the patient and her parents was performed. The analysis focused on recessive model including compound heterozygous mutations. Sanger sequencing was used to validate identified mutations and to screen the patient's newborn sister and grandparents. Expression and localization analysis were performed in the patient's duodenal biopsies using immunohistochemistry. RESULTS: Using WES we identified a new compound heterozygote mutation in sucrase-isomaltase (SI) gene; a maternal inherited known V577G mutation, and a novel paternal inherited C1531W mutation. Importantly, the newborn offspring carried similar compound heterozygous mutations. Computational predictions suggest that both mutations highly destabilize the protein. SI expression and localization studies determined that the mutated SI protein was not expressed on the brush border membrane in the patient's duodenal biopsies, verifying the diagnosis of congenital sucrase-isomaltase deficiency (CSID). CONCLUSIONS: The novel compound heterozygote V577G/C1531W SI mutations lead to lack of SI expression in the duodenal brush border, confirming the diagnosis of CSID. These cases of CSID extend the molecular spectrum of this condition, further directing a more adequate dietary intervention for the patient and newborn sibling.

MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder.

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285∗), c.247_250del (p.Asn83Hisfs∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

A Novel Mutation in a Critical Region for the Methyl Donor Binding in DNMT3B Causes Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome (ICF).

PURPOSE: Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is an extremely rare autosomal recessive disease. The immune phenotype is characterized by hypogammaglobulinemia in the presence of B cells. T cell lymphopenia also develops in some patients. We sought to further investigate the immune defect in an ICF patient with a novel missense mutation in DNMT3B and a severe phenotype. METHODS: Patient lymphocytes were examined for subset counts, immunoglobulin levels, T and B cell de novo production (via excision circles) and receptor repertoire diversity. Mutated DNMT3B protein structure was modeled to assess the effect of a mutation located outside of the catalytic region on protein function. RESULTS: A novel homozygous missense mutation, Ala585Thr, was found in DNMT3B. The patient had decreased B cell counts with hypogammaglobulinemia, and normal T cell counts. CD4+ T cells decreased over time, leading to an inversion of the CD4+ to CD8+ ratio. Excision circle copy numbers were normal, signifying normal de novo lymphocyte production, but the ratio between naïve and total B cells was low, indicating decreased in vivo B cell replication. T and B cell receptor repertoires displayed normal diversity. Computerized modeling of the mutated Ala585 residue suggested reduced thermostability, possibly affecting the enzyme kinetics. CONCLUSIONS: Our results highlight the existence of a T cell defect that develops over time in ICF patient, in addition to the known B cell dysfunction. With intravenous immunoglobulin (IVIG) treatment ameliorating the B cell defect, the extent of CD4+ lymphopenia may determine the severity of ICF immunodeficiency.

G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures.

BACKGROUND: Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates. RESULTS: We present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D . CONCLUSIONS: G23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.

Loss of function of NaPiIIa causes nephrocalcinosis and possibly kidney insufficiency.

BACKGROUND: Inherited metabolic disorders associated with nephrocalcinosis are rare conditions. The aim of this study was to identify the genetic cause of an Israeli-Arab boy from a consanguineous family with severe nephrocalcinosis and kidney insufficiency. METHODS: Clinical and biochemical data of the proband and family members were obtained from both previous and recent medical charts. Genomic DNA was isolated from peripheral blood cells. The coding sequence and splice sites of candidate genes (CYP24A1, CYP27B1, FGF23, KLOTHO, SLC34A3 and SLC34A1) were sequenced directly. Functional studies were performed in Xenopus laevis oocytes and in transfected opossum kidney (OK) cells. RESULTS: Our patient was identified as having nephrocalcinosis in utero, and at the age of 16.5 years, he had kidney insufficiency but no bone disease. Genetic analysis revealed a novel homozygous missense mutation, Arg215Gln, in SLC34A1, which encodes the renal sodium phosphate cotransporter NaPiIIa. Functional studies of the Arg215Gln mutant revealed reduced transport activity in Xenopus laevis oocytes and increased intracellular cytoplasmic accumulation in OK cells. CONCLUSIONS: Our findings show that dysfunction of the human NaPiIIa causes severe renal calcification that may eventually lead to reduced kidney function, rather than complications of phosphate loss.

Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects.

The analysis of individuals with telomere defects may shed light on the delicate interplay of factors controlling genome stability, premature aging, and cancer. We herein describe two Coats plus patients with telomere and genomic defects; both harbor distinct, novel mutations in STN1, a member of the human CTC1-STN1-TEN1 (CST) complex, thus linking this gene for the first time to a human telomeropathy. We characterized the patients' phenotype, recapitulated it in a zebrafish model and rescued cellular and clinical aspects by the ectopic expression of wild-type STN1 or by thalidomide treatment. Interestingly, a significant lengthy control of the gastrointestinal bleeding in one of our patients was achieved by thalidomide treatment, exemplifying a successful bed-to-bench-and-back approach.

Expanding the molecular diversity and phenotypic spectrum of glycerol 3-phosphate dehydrogenase 1 deficiency.

Transient infantile hypertriglyceridemia (HTGT1; OMIM #614480) is a rare autosomal recessive disorder, which manifests in early infancy with transient hypertriglyceridemia, hepatomegaly, elevated liver enzymes, persistent fatty liver and hepatic fibrosis. This rare clinical entity is caused by inactivating mutations in the GPD1 gene, which encodes the cytosolic isoform of glycerol-3-phosphate dehydrogenase. Here we report on four patients from three unrelated families of diverse ethnic origins, who presented with hepatomegaly, liver steatosis, hypertriglyceridemia, with or without fasting ketotic hypoglycemia. Whole exome sequencing revealed the affected individuals to harbor deleterious biallelic mutations in the GPD1 gene, including the previously undescribed c.806G > A (p.Arg269Gln) and c.640T > C (p.Cys214Arg) mutations. The clinical features in three of our patients showed several differences compared to the original reports. One subject presented with recurrent episodes of fasting hypoglycemia along with hepatomegaly, hypetriglyceridemia, and elevated liver enzymes; the second showed a severe liver disease, with intrahepatic cholestasis associated with kidney involvement; finally, the third presented persistent hypertriglyceridemia at the age of 30 years. These findings expand the current knowledge of this rare disorder, both with regard to the phenotype and molecular basis. The enlarged phenotypic spectrum of glycerol-3-phosphate dehydrogenase 1 deficiency can mimic other inborn errors of metabolism with liver involvement and should alert clinicians to recognize this entity by considering GPD1 mutations in appropriate clinical settings.

e23D: database and visualization of A-to-I RNA editing sites mapped to 3D protein structures.

UNLABELLED: e23D, a database of A-to-I RNA editing sites from human, mouse and fly mapped to evolutionary related protein 3D structures, is presented. Genomic coordinates of A-to-I RNA editing sites are converted to protein coordinates and mapped onto 3D structures from PDB or theoretical models from ModBase. e23D allows visualization of the protein structure, modeling of recoding events and orientation of the editing with respect to nearby genomic functional sites from databases of disease causing mutations and genomic polymorphism. AVAILABILITY AND IMPLEMENTATION: http://www.sheba-cancer.org.il/e23D CONTACT: oz.solomon@live.biu.ac.il or Eran.Eyal@sheba.health.gov.il.

The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.