Genetic Markers Among the Israeli Druze Minority Population With End-Stage Kidney Disease.

RATIONALE & OBJECTIVE: Genetic etiologies have been identified among approximately 10% of adults with chronic kidney disease (CKD). However, data are lacking regarding the prevalence of monogenic etiologies especially among members of minority groups. This study characterized the genetic markers among members of an Israeli minority group with end-stage kidney disease (ESKD). STUDY DESIGN: A national-multicenter cross-sectional study of Israeli Druze patients (an Arabic-speaking Near-Eastern transnational population isolate) who are receiving maintenance dialysis for ESKD. All study participants underwent exome sequencing. SETTING & PARTICIPANTS: We recruited 94 adults with ESKD, comprising 97% of the total 97 Druze individuals throughout Israel being treated with dialysis during the study period. PREDICTORS: Demographics and clinical characteristics of kidney disease. OUTCOME: Genetic markers. ANALYTICAL APPROACH: Whole-exome sequencing and the relationship of markers to clinical phenotypes. RESULTS: We identified genetic etiologies in 17 of 94 participants (18%). None had a previous molecular diagnosis. A novel, population-specific, WDR19 homozygous pathogenic variant (p.Cys293Tyr) was the most common genetic finding. Other monogenic etiologies included PKD1, PKD2, type IV collagen mutations, and monogenic forms of noncommunicable diseases. The pre-exome clinical diagnosis corresponded to the final molecular diagnosis in fewer than half of the participants. LIMITATIONS: This study was limited to Druze individuals, so its generalizability may be limited. CONCLUSIONS: Exome sequencing identified a genetic diagnosis in approximately 18% of Druze individuals with ESKD. These results support conducting genetic analyses in minority populations with high rates of CKD and for whom phenotypic disease specificity may be low. PLAIN-LANGUAGE SUMMARY: Chronic kidney disease (CKD) affects many people worldwide and has multiple genetic causes. However, there is limited information on the prevalence of genetic etiologies, especially among minority populations. Our national-multicenter study focused on Israeli Druze patients. Using exome-sequencing, we identified previously undetected genetic causes in nearly 20% of patients, including a new and population-specific WDR19 homozygous pathogenic variant. This mutation has not been previously described; it is extremely rare globally but is common among the Druze, which highlights the importance of studying minority populations with high rates of CKD. Our findings provide insights into the genetic basis of end-stage kidney disease in the Israeli Druze, expand the WDR19 phenotypic spectrum, and emphasize the potential value of genetic testing in such populations.

Reassessing the role of high dose cytarabine and mitoxantrone in relapsed/refractory acute myeloid leukemia.

A substantial segment of patients with acute myeloid leukemia (AML) will relapse following an initial response to induction therapy or will prove to be primary refractory. High-dose cytarabine and mitoxantrone (HiDAC/MITO) is an established salvage therapy for these patients. We studied all adult patients with relapsed/refractory (R/R) AML who were treated with HiDAC/MITO in our center between the years 2008-2017. To determine whether responding patients harbored a unique molecular signature, we performed targeted next-generation sequencing (NGS) on a subset of patients. The study cohort consisted of 172 patients with a median age of 54 years (range 18-77). The composite complete remission rate was 58%; 11 patients (6%) died during salvage therapy. Median survival was 11.4 months with a 1-year survival rate of 48%. In multivariate analysis favorable risk cytogenetics [Odds ratio (OR)=0.34, confidence interval (CI) 95%, 0.17-0.68; P = 0.002], and de-novo AML (OR = 0.4, CI 95%, 0.16-0.98; P = 0.047) were independently associated with a favorable response. Patients who attained a complete remission had a median survival of 43.7 months compared with 5.2 months for refractory patients (p < 0.0001). Neither the FLT3-ITD and NPM1 mutational status nor the indication for salvage therapy significantly impacted on the response to HiDAC/MITO salvage. NGS analysis identified 20 different mutations across the myeloid gene spectrum with a distinct TP53 signature detected in non-responding patients. HiDAC/MITO is an effective salvage regimen in R/R AML, however patients with adverse cytogenetics or secondary disease may not benefit as much from this approach.

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.

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.

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.

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure.

Analysis of hematopoietic stem cells (HSCs) in factor VIII knockout (FVIIIKO) mice revealed a novel regulatory role for the coagulation cascade in hematopoiesis. Thus, HSCs in FVIIIKO mice had reduced proportions of CD34(low) cells within Lin(-)Sca(+)Kit(+) progenitors, and exhibited reduced long-term repopulating capacity as well as hyper granulocyte-colony-stimulating factor (G-CSF)-induced mobilization. This disregulation of HSCs is likely caused by reduced levels of thrombin, and is associated with altered protease-activated receptor 1 (PAR1) signaling, as PAR1 KO mice also exhibited enhanced G-CSF-induced mobilization. Analysis of reciprocal bone marrow (BM) chimera (FVIIIKO BM into wild-type recipients and vice versa) and the detection of PAR1 expression on stromal elements indicates that this phenotype is likely controlled by stromal elements. Micro-computed tomography analysis of distal tibia metaphyses also revealed for the first time a major impact of the FVIII/thrombin/PAR1 axis on the dynamic bone structure, showing reduced bone:tissue volume ratio and trabecular number in FVIIIKO and PAR1KO mice. Taken together, these results show a critical and novel role for the coagulation cascade, mediated in part by thrombin-PAR1 interaction, and regulates HSC maintenance and a reciprocal interplay between HSCs and the dynamic bone structure.

Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors.

Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

Nanog inhibits the switch of myogenic cells towards the osteogenic lineage.

The homeodomain transcription factor Nanog has been implicated in inhibiting differentiation and controlling pluripotency of embryonic stem (ES) cells. We used ectopic expression of Nanog in the myogenic committed C2 cells to dissect these properties. Expression of Nanog in C2 cells does not alter terminal muscle differentiation but has a profound effect on their switch to differentiate along the osteogenic lineage upon BMP treatment. Gene expression profiling revealed that ERK 1/2 phosphorylation, alkaline-phosphatase activity and osteocalcin expression were induced to much lower extent and remained suppressed even after 96h. in Nanog expressing C2 cells, compared to control C2 cells. Hence, Nanog does not inhibit terminal differentiation of committed cells but it is an inhibitor of trans-differentiation that is dependent on de-novo activation of gene transcription.

CD24 is a new oncogene, early at the multistep process of colorectal cancer carcinogenesis.

BACKGROUND & AIMS: The aim of this study was to identify genes that play a role in colorectal cancer (CRC) carcinogenesis by analysis of differential gene expression of normal and transformed CRC cell lines. METHODS: Gene expression array analysis ([RG-U34] GeneChip) was performed in normal and transformed rat intestinal epithelial cells before and after exposures to celecoxib. In particular, we were looking for (1) altered gene expression in the transformed cells that reverts to normal following exposure to a selective cyclooxygenase-2 inhibitor, (2) novel genes, and (3) genes encoding membrane receptors or ligands. As a validation of the results and for human patients, immunohistochemistry was performed on 398 biological samples from the gastrointestinal tract (normal, polyps, and adenocarcinomas). Human cancer cell lines were tested for their response to anti-CD24 monoclonal antibodies. RESULTS: A total of 1081 genes were differently expressed following malignant transformation; 71 genes showed altered expression that reverted to normal following treatment with celecoxib, including the CD24 gene. Immunohistochemistry confirmed that increased expression of CD24 is an early event in CRC carcinogenesis. It was expressed in 90.7% of adenomas and 86.3% of CRCs. Very low expression was seen in normal epithelium (16.6%). Human cancer cell lines showed growth inhibition in response to the antibodies, according to their expression levels of CD24 and in dose- and time-dependent manners. These results were repetitive for 3 different antibodies. CONCLUSIONS: CD24 is overexpressed in the colonic mucosa, already at an early stage of carcinogenesis. It may be a useful target for early detection and in CRC therapy.

Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the Mdr2-knockout mice.

Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model.

Evolution of a T-B- SCID into an Omenn syndrome phenotype following parainfluenza 3 virus infection.

Mutations in both of the recombination activating genes (RAG)1 and RAG2 can lead to either T-B-severe combined immune deficiency (SCID) or Omenn syndrome (OS), two diseases presenting with totally different clinical and laboratory manifestations. The fact that the same mutations can cause either T-B- SCID or OS, even within the same family, lends credibility to the hypothesis that an additional factor (autoantigen or exoantigen) is required in certain circumstances for the development of OS phenotype. We investigated three patients from the same extended family who presented as T-B- SCID due to a homozygous mutation (G1305T) in the RAG2 gene. Our data support the notion that mutated RAG proteins may not always be sufficient to cause OS phenotype, and show evolution from a T-B- SCID into a typical OS phenotype subsequent to parainfluenza 3 virus infection.