Publications by authors named "David A Zeevi"

20 Publications

  • Page 1 of 1

When a Root Is the Cause of Infection.

World Neurosurg 2020 Dec 2;144:258-261.e1. Epub 2020 Sep 2.

Department of Neurosurgery, Shaare Zedek Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, Israel. Electronic address:

Background: Sinorhizobium meliloti is a phytobacterium found in the root nodules of plants, where it is involved in fixing nitrogen for delivery to the roots in exchange for a photosynthate carbon source. There have been no reported cases of S. meliloti infection in humans. We conducted a retrospective review of clinical records and diagnostic tests.

Case Description: An 81-year-old woman who presented to the emergency department with a 1-day history of progressive decline in her level of consciousness following a head injury and deep scalp laceration. Her medical history was significant for a ventriculoperitoneal shunt due to normal pressure hydrocephalus. Imaging studies revealed hydrocephalus and a tear in the shunt catheter. Cerebrospinal fluid analysis was not suggestive for meningitis. Cerebrospinal fluid culture revealed an unfamiliar organism, identified as S. meliloti following sequencing of its entire genome, which was considered a contaminant. The patient subsequently developed peritonitis, and the same pathogen was detected in the peritoneal fluid, suggesting distal shunt infection. Symptoms resolved after shunt removal and antibiotic treatment. Thorough history taking revealed that the patient had fallen and struck her head against a flowerpot.

Conclusions: S. meliloti is a phytopathogen that should not be easily disregarded as a contaminant when isolated from human sterile fluids or tissues. Aggressive management including removal of infected hardware, if present, is required to ensure resolution of infection. It emphasizes the importance of thorough history taking.
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http://dx.doi.org/10.1016/j.wneu.2020.08.174DOI Listing
December 2020

The utility of MALDI-TOF MS for outbreak investigation in the neonatal intensive care unit.

Eur J Pediatr 2020 Dec 10;179(12):1843-1849. Epub 2020 Jun 10.

Faculty of Medicine, Hebrew University, Jerusalem, Israel.

Our aim was to evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), routinely used in the microbiology laboratory for bacterial identification, for bacterial typing in the setting of extended spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KP) outbreak in the neonatal intensive care unit (NICU). Isolates from a 2011 outbreak in the NICU were retrieved from frozen stocks and analyzed by MALDI-TOF. The MALDI typing was compared with core genome multilocus sequence typing (cg-MLST). MALDI typing divided the 33 outbreak isolates into 2 clones: sequence type (ST)-290 and 405. These results were in complete agreement with cg-MLST results. The differentiation of the outbreak isolates into two clones correlated with the patients' location in the NICU, but also with their place of residence.Conclusion: Here, we show that MALDI-TOF MS, which has been integrated into the microbiology laboratory workflow for microbial species identification, can be secondarily used for epidemiological typing at no added cost. What is Known: • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now routinely used in the microbiology laboratory for bacterial identification What is New: • MALDI typing was used for outbreak investigation in the NICU and divided the outbreak isolates into two clones • MALDI-TOF MS may be secondarily used for epidemiological typing at no added cost.
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http://dx.doi.org/10.1007/s00431-020-03696-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283987PMC
December 2020

The decision-making process, experience, and perceptions of preimplantation genetic testing (PGT) users.

J Assist Reprod Genet 2020 Aug 27;37(8):1903-1912. Epub 2020 May 27.

Shaare Zedek Medical Center- Medical Genetics Institute, Jerusalem, Israel.

Purpose: The decision to undergo preimplantation genetic testing (PGT) entails a variety of personal and societal variables. Although PGT technology is widely accepted and used, few studies have queried the motives and concerns of PGT users; moreover, in-depth qualitative data regarding the PGT experience is scant.

Methods: In order to explore and analyze the experience, concerns, expectations, and attitudes toward the PGT technique and its implications, semi-structured interviews were conducted in a single tertiary medical center with 43 Israeli PGT users for HLA matching and autosomal dominant, autosomal recessive, and X-linked genetic disorders.

Results: The primary considerations in choosing PGT were prevention of birth of a child who would suffer a terminal or chronic disease as well as abrogation of a familial genetic condition. Religion played a decisive role in accepting PGT as an antenatal option. Regarding satisfaction with the PGT experience, many interviewees highlighted the need for greater attention to be given to potential stages of failure throughout the procedure and the need for emotional support. Our clinical results regarding implantation rate and cumulative live birth rate are 38-40% and 27-30%, respectively.

Conclusion: This survey broadens understanding of the specialized needs of women, couples, and minority groups undergoing PGT and underscores the relevance of counseling services for PGT users.
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http://dx.doi.org/10.1007/s10815-020-01840-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468006PMC
August 2020

Bi-allelic Loss-of-Function Variants in NUP188 Cause a Recognizable Syndrome Characterized by Neurologic, Ocular, and Cardiac Abnormalities.

Am J Hum Genet 2020 05 9;106(5):623-631. Epub 2020 Apr 9.

Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, WA 98195, USA; Seattle Children's Hospital, Seattle, WA 98105, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA 98195, USA. Electronic address:

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.
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http://dx.doi.org/10.1016/j.ajhg.2020.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212259PMC
May 2020

A founder deletion in the gene associated with congenital stationary night blindness and myopia is highly prevalent in Ashkenazi Jews.

Hum Genome Var 2019 12;6:45. Epub 2019 Sep 12.

Dor Yeshorim, The Committee for Prevention of Jewish Genetic Diseases, Brooklyn, NY USA.

Congenital stationary night blindness (CSNB) is a disease affecting the night vision of individuals. Previous studies identified as a gene involved in reduced night vision. Homozygous deletion of was the cause of CSNB in several children in 6 Ashkenazi Jewish families, thereby prompting further investigation of the carrier status within the families as well as in large cohorts of unrelated Ashkenazi and Sephardi individuals. Affected children were tested with a CSNB next-generation (NextGen) sequencing panel. A deletion of exons 2 through 7 was detected and confirmed by PCR and sequence analysis. A TaqMan-based assay was used to assess the frequency of this deletion in 18266 individuals of Jewish descent. High-throughput amplicon sequencing was performed on 380 samples to determine the putative deletion-flanking founder haplotype. Heterozygous deletions were found in 2.75% (1/36) of Ashkenazi subjects and in 1.22% (1/82) individuals of mixed Ashkenazi/Sephardic origin. The homozygous deletion frequency in our data was 0.03% (1/4025) and was only found in Ashkenazi Jewish individuals. Homozygous deletion of exons 2-7 in is a common cause of CSNB and myopia in many Ashkenazi Jewish patients. This deletion is a founder Ashkenazi Jewish deletion.
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http://dx.doi.org/10.1038/s41439-019-0076-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804618PMC
September 2019

A Cluster of Bacillus cereus Infections in the Neonatal Intensive Care Unit: Epidemiologic and Whole-genome Sequencing Analysis.

Pediatr Infect Dis J 2019 11;38(11):e301-e306

Faculty of Medicine, Hebrew University.

Bacillus cereus isolates causing an outbreak in the neonatal intensive care unit were investigated using whole-genome sequencing. The outbreak coincided with construction work performed adjacent to the neonatal intensive care unit and ceased after strict sealing of the construction area. We found the outbreak to be polyclonal, however, the clonality did not correlate with the virulence in vivo. Genotypically similar isolates were associated with both lethal/severe infection and colonization/environmental contamination. Environmental bacterial load may be a major determinant of infection, especially in high-risk patients. Clinicians should be alert to unusual increase in B. cereus isolations from clinical cultures to facilitate early recognition and investigations of Bacillus outbreaks and pseudo-outbreaks. The integration of genomics into the classical infectious disease work can augment our understanding of pathogen transmission and virulence, and can rapidly assist our response to unusual disease trends.
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http://dx.doi.org/10.1097/INF.0000000000002441DOI Listing
November 2019

Study of carrier frequency of Warsaw breakage syndrome in the Ashkenazi Jewish population and presentation of two cases.

Am J Med Genet A 2019 10 9;179(10):2144-2151. Epub 2019 Jul 9.

Clinical Genetic Services, Department of Pediatrics, NYU School of Medicine, New York, New York.

Warsaw breakage syndrome (WABS), caused by bi-allelic variants in the DDX11 gene, is a rare cohesinopathy characterized by pre- and postnatal growth retardation, microcephaly, intellectual disability, facial dysmorphia, and sensorineural hearing loss due to cochlear hypoplasia. The DDX11 gene codes for an iron-sulfur DNA helicase in the Superfamily 2 helicases and plays an important role in genomic stability and maintenance. Fourteen individuals with WABS have been previously reported in the medical literature. Affected individuals have been of various ethnic backgrounds with different pathogenic variants. We report two unrelated individuals of Ashkenazi Jewish descent affected with WABS, who are homozygous for the c.1763-1G>C variant in the DDX11 gene. Their phenotype is consistent with previously reported individuals. RNA studies showed that this variant causes an alternative splice acceptor site leading to a frameshift in the open reading frame. Carrier screening of the c.1763-1G>C variant in the Jewish population revealed a high carrier frequency of 1 in 68 in the Ashkenazi Jewish population. Due to the high carrier frequency and the low number of affected individuals, we hypothesize a high rate of miscarriage of homozygous fetuses and/or subfertility for carrier couples. If the carrier frequency is reproducible in additional Ashkenazi Jewish populations, we suggest including DDX11 to Ashkenazi Jewish carrier screening panels.
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http://dx.doi.org/10.1002/ajmg.a.61284DOI Listing
October 2019

Off the street phasing (OTSP): no hassle haplotype phasing for molecular PGD applications.

J Assist Reprod Genet 2019 Apr 8;36(4):727-739. Epub 2019 Jan 8.

Medical Genetics Institute, Shaare Zedek Medical Center (SZMC), Bayit Str. 12, P.O.Box 3235, 91031, Jerusalem, Israel.

Purpose: Pre-implantation genetic diagnosis (PGD) for molecular disorders requires the construction of parental haplotypes. Classically, haplotype resolution ("phasing") is obtained by genotyping multiple polymorphic markers in both parents and at least one additional relative. However, this process is time-consuming, and immediate family members are not always available. The recent availability of massive genomic data for many populations promises to eliminate the needs for developing family-specific assays and for recruiting additional family members. In this study, we aimed to validate population-assisted haplotype phasing for PGD.

Methods: Targeted sequencing of CFTR gene variants and ~ 1700 flanking polymorphic SNPs (± 2 Mb) was performed on 54 individuals from 12 PGD families of (a) Full Ashkenazi (FA; n = 16), (b) mixed Ashkenazi (MA; n = 23 individuals with at least one Ashkenazi and one non-Ashkenazi grandparents), or (c) non-Ashkenazi (NA; n = 15) descent. Heterozygous genotype calls in each individual were phased using various whole genome reference panels and appropriate computational models. All computationally derived haplotype predictions were benchmarked against trio-based phasing.

Results: Using the Ashkenazi reference panel, phasing of FA was highly accurate (99.4% ± 0.2% accuracy); phasing of MA was less accurate (95.4% ± 4.5% accuracy); and phasing of NA was predictably low (83.4% ± 6.6% accuracy). Strikingly, for founder mutation carriers, our haplotyping approach facilitated near perfect phasing accuracy (99.9% ± 0.1% and 98.2% ± 2.8% accuracy for W1282X and delF508 carriers, respectively).

Conclusions: Our results demonstrate the feasibility of replacing classical haplotype phasing with population-based phasing with uncompromised accuracy.
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http://dx.doi.org/10.1007/s10815-018-1392-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504987PMC
April 2019

Haploseek: a 24-hour all-in-one method for preimplantation genetic diagnosis (PGD) of monogenic disease and aneuploidy.

Genet Med 2019 06 19;21(6):1390-1399. Epub 2018 Nov 19.

Translational Genomics Lab, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.

Purpose: To develop an economical, user-friendly, and accurate all-in-one next-generation sequencing (NGS)-based workflow for single-cell gene variant detection combined with comprehensive chromosome screening in a 24-hour workflow protocol.

Methods: We subjected single lymphoblast cells or blastomere/blastocyst biopsies from four different families to low coverage (0.3×-1.4×) genome sequencing. We combined copy-number variant (CNV) detection and whole-genome haplotype phase prediction via Haploseek, a novel, user-friendly analysis pipeline. We validated haplotype predictions for each sample by comparing with clinical preimplantation genetic diagnosis (PGD) case results or by single-nucleotide polymorphism (SNP) microarray analysis of bulk DNA from each respective lymphoblast culture donor. CNV predictions were validated by established commercial kits for single-cell CNV prediction.

Results: Haplotype phasing of the single lymphoblast/embryo biopsy sequencing data was highly concordant with relevant ground truth haplotypes in all samples/biopsies from all four families. In addition, whole-genome copy-number assessments were concordant with the results of a commercial kit.

Conclusion: Our results demonstrate the establishment of a reliable method for all-in-one molecular and chromosomal diagnosis of single cells. Important features of the Haploseek pipeline include rapid sample processing, rapid sequencing, streamlined analysis, and user-friendly reporting, so as to expedite clinical PGD implementation.
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http://dx.doi.org/10.1038/s41436-018-0351-7DOI Listing
June 2019

Noninvasive paternal exclusion testing for cystic fibrosis in the first five to eight weeks of gestation.

Sci Rep 2018 10 29;8(1):15941. Epub 2018 Oct 29.

Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.

Prenatal genetic testing is not generally applicable to the very early stages of pregnancy (prior to week 8 gestation), a time period that is crucial to pregnant couples with high risk for transmission of genetic disease to their fetus. Therefore, we developed a new ultra-sensitive targeted next generation sequencing method for noninvasive haplotype-based paternal allele exclusion testing of the cystic fibrosis-associated gene, CFTR. This new method was compared to a conventional library prep and sequencing analysis method and all test results were validated by amniotic fluid testing at later stages of pregnancy. Out of 7 enrolled couples, who provided at least two blood samples (at least one week apart) for noninvasive CFTR testing, a result was obtained for 6 fetuses. Using the new hypersensitive method, all six couples (100%) received a correct diagnosis for the paternal allele as opposed to 3/6 (50%) when tested with the conventional strategy. Among 4 couples who provided just one early pregnancy blood draw for analysis, diagnosis was possible in one fetus, but only using the ultra-sensitive method. Thus, we describe a novel noninvasive CFTR screening method which demonstrates unprecedented fetal allele typing accuracy in the earliest stages of pregnancy.
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http://dx.doi.org/10.1038/s41598-018-34396-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205998PMC
October 2018

Acceptable applications of preimplantation genetic diagnosis (PGD) among Israeli PGD users.

Eur J Hum Genet 2017 10 26;25(10):1113-1117. Epub 2017 Jul 26.

Medical Genetics Institute - Shaare Zedek Medical Center, Hebrew University Hadassah Medical School, Jerusalem, Israel.

The use of PGD technology to select against genetic disorders and traits is increasing. Although PGD may eliminate some of the obstacles related to conservative options of prenatal diagnosis, it can raise personal, social and moral questions. Ethical issues concerning the justified uses of PGD are a subject of ongoing debate among medical and bioethical communities. Although attitudes toward the acceptable uses of PGD were evaluated among population groups worldwide, bioethics councils were criticized for ignoring public perspectives. In the last decade PGD has been widely used in Israel. The ethical guidelines were created solely by medical-bioethics experts and, some felt, totally isolated from public opinions. Semi-structured in-depth interviews of 37 users (carriers of autosomal recessive, dominant and X-linked disorders, and HLA-matching) were performed. The interviews explored attitudes toward ethical and sociological aspects of PGD. The overall results of this study show highly favorable attitudes of Israeli PGD users toward medical applications. Furthermore, our subjects demonstrate a more permissive stand toward the controversial application of social sex selection albeit with strong objection to esthetic means of selection. PGD users are coping with both genetic disease and load of the PGD procedure. Taking into consideration their opinion is important since it reflects the gains and burdens of these procedures alongside the demand for future optional services. Their attitudes should play an important role in the professional discussion concerning the justified uses of PGD and should significantly influence the design of policy making in this field.
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http://dx.doi.org/10.1038/ejhg.2017.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602014PMC
October 2017

Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations.

J Clin Invest 2015 Oct 31;125(10):3757-65. Epub 2015 Aug 31.

Background: Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population.

Methods: Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease-associated founder mutation-flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods.

Results: Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation-flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles.

Conclusions: The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations.

Funding: SZMC, Protalix Biotherapeutics Inc., and Centogene AG.
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http://dx.doi.org/10.1172/JCI79322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607112PMC
October 2015

TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

PLoS One 2015 31;10(7):e0134120. Epub 2015 Jul 31.

Human Genetics, Hebrew University Medical School, Jerusalem, Israel; Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.

Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134120PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521930PMC
May 2016

An intronic haplotype in α galactosidase A is associated with reduced mRNA expression in males with cryptogenic stroke.

Gene 2014 Oct 4;549(2):275-9. Epub 2014 Aug 4.

Medical Genetics Institute, Shaare Zedek Medical Center, Affiliated with the Hadassah-Hebrew University School of Medicine, Jerusalem, Israel.

Persons with unexplained early-onset stroke have been targeted for screening surveys for Fabry disease, the most common of the three X-linked lysosomal disorders, because Fabry patients with stroke are more likely to have the life-threatening progressive cardiac and renal manifestations and would therefore most benefit from early diagnosis and intervention with enzyme replacement therapy (ERT). Among 175 Israeli patients with unexplained cryptogenic stroke screened for mutations in the Fabry α galactosidase A (GLA) gene, sequencing identified six with 2-4 GLA intronic variants, one of whose father and three sisters had the same variants. Two variants, c.640-16A>G (g.10115A>G) in intron 4 and c.1000-22C>T (g.10956C>T) in intron 6, were common to all patients. However, three males with a common four variant intronic haplotype had low residual enzyme activity and ~50% reduced mRNA expression. Transcript splice-site defects were not identified in any of the index cases and X-chromosome inactivation was not highly skewed in the six females. These data do not suggest that GLA intronic variants, per se, are pathogenic. Nonetheless, it is clear that a certain intronic haplotype in males with cryptogenic stroke is associated with reduced GLA expression and function.
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http://dx.doi.org/10.1016/j.gene.2014.08.004DOI Listing
October 2014

Preimplantation genetic diagnosis in genomic regions with duplications and pseudogenes: long-range PCR in the single-cell assay.

Hum Mutat 2013 May 19;34(5):792-9. Epub 2013 Mar 19.

Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.

Long-range PCR is generally employed for the analysis of disease-causing mutations in genes with homologous pseudogene copies. However, long-range PCR is challenging when performed on single cells, as in preimplantation genetic diagnosis (PGD) of monogenic disorders. PGD on single cells requires concurrent analysis of a mutation together with multiple linked polymorphic markers from closely related family members to prevent misdiagnosis. In PGD cases involving childless de novo mutation carriers, linkage cannot be performed based on family members but rather must first be identified in single gametes. This can be an especially difficult task if the mutation to be assayed lies in a duplicated genomic region because gene-specific long-range PCR must be coupled with short-range PCR analysis of genetic markers on single cells. Here, we describe a novel method by which accurate PGD of pseudogene-homologous mutations can be achieved. Essentially, we performed whole genome amplification on single sperm or blastomeres followed by haplotype construction and long-range PCR-based mutation analysis. This original and universal strategy was used to establish allelic association for two different mutations in genes with one or more pseudogene copies (IKBKG and PKD1). The method was also sensitive enough to detect unexpected germline mosaicism in one mutation carrier.
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http://dx.doi.org/10.1002/humu.22298DOI Listing
May 2013

Mucolipidosis type IV and the mucolipins.

Biochem Soc Trans 2010 Dec;38(6):1432-5

Department of Human Genetics, Hadassah Hebrew University Hospital, Jerusalem 91120, Israel.

MLIV (mucolipidosis type IV) is a neurodegenerative lysosomal storage disorder caused by mutations in MCOLN1, a gene that encodes TRPML1 (mucolipin-1), a member of the TRPML (transient receptor potential mucolipin) cation channels. Two additional homologues are TRPML2 and TRPML3 comprising the TRPML subgroup in the TRP superfamily. The three proteins play apparently key roles along the endocytosis process, and thus their cellular localization varies among the different group members. Thus TRPML1 is localized exclusively to late endosomes and lysosomes, TRPML2 is primarily located in the recycling clathrin-independent GPI (glycosylphosphatidylinositol)-anchored proteins and early endosomes, and TRPML3 is primarily located in early endosomes. Apparently, all three proteins' main physiological function underlies Ca(2+) channelling, regulating the endocytosis process. Recent findings also indicate that the three TRPML proteins form heteromeric complexes at least in some of their cellular content. The physiological role of these complexes in lysosomal function remains to be elucidated, as well as their effect on the pathophysiology of MLIV. Another open question is whether any one of the TRPMLs bears additional function in channel activity.
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http://dx.doi.org/10.1042/BST0381432DOI Listing
December 2010

Heteromultimeric TRPML channel assemblies play a crucial role in the regulation of cell viability models and starvation-induced autophagy.

J Cell Sci 2010 Sep 24;123(Pt 18):3112-24. Epub 2010 Aug 24.

Monique and Jacques Roboh Department of Genetic Research, Institute of Medical Research Israel-Canada (IMRIC), Faculty of Medicine of the Hebrew University and Hadassah Hebrew University Hospital, Jerusalem 91120, Israel.

The mucolipin (TRPML) subfamily of transient receptor potential (TRP) cation channels consists of three members that play various roles in the regulation of membrane and protein sorting along endo-lysosomal pathways. Loss-of-function mutations in TRPML1 cause the neurodegenerative lysosomal storage disorder, mucolipidosis type IV (MLIV), whereas a gain-of-function mutation in TRPML3 is principally implicated in the hearing-impaired and abnormally pigmented varitint-waddler mouse. Currently, TRPML2 is not implicated in any pathological disorder, but we have recently shown that it is a functional cation channel that physically interacts with TRPML1 and TRPML3 to potentially regulate lysosomal integrity. Here, we show that mutant TRPMLs heteromultimerize with other mutant and wild-type TRPMLs to regulate cell viability and starvation-induced autophagy, a process that mediates macromolecular and organellar turnover under cell starvation conditions. Heteromultimerization of dominant-negative TRPMLs with constitutively active TRPMLs rescues cells from the cytotoxic effects of TRPML constitutive activity. Moreover, dominant-negative TRPML1 channels, including a mutant channel directly implicated in MLIV pathology, also inhibit starvation-induced autophagy by interacting with and affecting native TRPML channel function. Collectively, our results indicate that heteromultimerization of TRPML channels plays a role in various TRPML-regulated mechanisms.
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http://dx.doi.org/10.1242/jcs.067330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931605PMC
September 2010

Constitutive activity of the human TRPML2 channel induces cell degeneration.

J Biol Chem 2010 Jan 23;285(4):2771-82. Epub 2009 Nov 23.

Department of Medical Neurobiology and the Kühne Minerva Center for Studies of Visual Transduction, Faculty of Medicine of the Hebrew University, Jerusalem, Israel.

The mucolipin (TRPML) ion channel proteins represent a distinct subfamily of channel proteins within the transient receptor potential (TRP) superfamily of cation channels. Mucolipin 1, 2, and 3 (TRPML1, -2, and -3, respectively) are channel proteins that share high sequence homology with each other and homology in the transmembrane domain with other TRPs. Mutations in the TRPML1 protein are implicated in mucolipidosis type IV, whereas mutations in TRPML3 are found in the varitint-waddler mouse. The properties of the wild type TRPML2 channel are not well known. Here we show functional expression of the wild type human TRPML2 channel (h-TRPML2). The channel is functional at the plasma membrane and characterized by a significant inward rectification similar to other constitutively active TRPML mutant isoforms. The h-TRPML2 channel displays nonselective cation permeability, which is Ca(2+)-permeable and inhibited by low extracytosolic pH but not Ca(2+) regulated. In addition, constitutively active h-TRPML2 leads to cell death by causing Ca(2+) overload. Furthermore, we demonstrate by functional mutation analysis that h-TRPML2 shares similar characteristics and structural similarities with other TRPML channels that regulate the channel in a similar manner. Hence, in addition to overall structure, all three TRPML channels also share common modes of regulation.
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http://dx.doi.org/10.1074/jbc.M109.046508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807332PMC
January 2010

A potentially dynamic lysosomal role for the endogenous TRPML proteins.

J Pathol 2009 Oct;219(2):153-62

Department of Human Genetics, Hadassah Hebrew University Hospital, Jerusalem, Israel.

Lysosomal storage disorders (LSDs) constitute a diverse group of inherited diseases that result from lysosomal storage of compounds occurring in direct consequence to deficiencies of proteins implicated in proper lysosomal function. Pathology in the LSD mucolipidosis type IV (MLIV), is characterized by lysosomal storage of lipids together with water-soluble materials in cells from every tissue and organ of affected patients. Mutations in the mucolipin 1 (TRPML1) protein cause MLIV and TRPML1 has also been shown to interact with two of its paralogous proteins, mucolipin 2 (TRPML2) and mucolipin 3 (TRPML3), in heterologous expression systems. Heterogeneous lysosomal storage is readily identified in electron micrographs of MLIV patient cells, suggesting that proper TRPML1 function is essential for the maintenance of lysosomal integrity. In order to investigate whether TRPML2 and TRPML3 also play a role in the maintenance of lysosomal integrity, we conducted gene-specific knockdown assays against these protein targets. Ultrastructural analysis revealed lysosomal inclusions in both TRPML2 and TRPML3 knockdown cells, suggestive of a common mechanism for these proteins, in parallel with TRPML1, in the regulation of lysosomal integrity. However, co-immunoprecipitation assays revealed that physical interactions between each of the endogenous TRPML proteins are quite limited. In addition, we found that all three endogenous proteins only partially co-localize with each other in lysosomal as well as extra-lysosomal compartments. This suggests that native TRPML2 and TRPML3 might participate with native TRPML1 in a dynamic form of lysosomal regulation. Given that depletion of TRPML2/3 led to lysosomal storage typical to an LSD, we propose that depletion of these proteins might also underlie novel LSD pathologies not described hitherto.
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http://dx.doi.org/10.1002/path.2587DOI Listing
October 2009

TRPML and lysosomal function.

Biochim Biophys Acta 2007 Aug 19;1772(8):851-8. Epub 2007 Jan 19.

Department of Human Genetics, Hadassah Hebrew University Hospital, Jerusalem, Israel.

Mucolipin 1 (MLN1), also known as TRPML1, is a member of the mucolipin family. The mucolipins are the only lysosomal proteins within the TRP superfamily. Mutations in the gene coding for TRPML1 result in a lysosomal storage disorder (LSD). This review summarizes the current knowledge related to this protein and the rest of the mucolipin family.
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http://dx.doi.org/10.1016/j.bbadis.2007.01.004DOI Listing
August 2007