Replication analysis confirms the association of ARID5B with childhood B-cell acute lymphoblastic leukemia

Although childhood acute lymphoblastic leukemia is the most common pediatric cancer, its etiology remains poorly understood. In an attempt to replicate the findings of 2 recent genome-wide association studies in a French-Canadian cohort, we confirmed the association of 5 SNPs [rs7073837 (P=4.2 × 10⁻⁴), rs10994982 (P=3.8 × 10⁻⁴), rs10740055 (P=1.6 × 10⁻⁵), rs10821936 (P=1.7 × 10⁻⁷) and rs7089424 (P=3.6 × 10⁻⁷)] in the ARID5B gene with childhood acute lymphoblastic leukemia. We also confirmed a selective effect for B-cell acute lymphoblastic leukemia with hyperdiploidy and report a putative gender-specific effect of ARID5B SNPs on acute lymphoblastic leukemia risk in males. This study provides a strong rationale for more detailed analysis to identify the causal variants at this locus and to better understand the overall functional contribution of ARID5B to childhood acute lymphoblastic leukemia susceptibility.     

Antioxidant and anti-inflammatory effects of mild hypothermia in the attenuation of liver injury due to azoxymethane toxicity in the mouse

Previous studies have demonstrated protective effects of mild hypothermia following acetaminophen (APAP)-induced acute liver failure (ALF). However, effects of this treatment in ALF due to other toxins have not yet been fully investigated. In the present study, the effects of mild hypothermia in relation to liver pathology, hepatic and cerebral glutathione, plasma ammonia concentrations, progression of encephalopathy, cerebral edema, and plasma proinflammatory cytokines were assessed in mice with ALF resulting from azoxymethane (AOM) hepatotoxicity, a well characterized model of toxic liver injury. Male C57BL/6 mice were treated with AOM (100 μg/g; i.p.) or saline and sacrificed at coma stages of encephalopathy in parallel with AOM mice maintained mildly hypothermic (35°C). AOM treatment led to hepatic damage, significant increase in plasma transaminase activity, decreased hepatic glutathione levels, and brain GSH/GSSG ratios as well as selective increases in expression of plasma proinflammatory cytokines. Mild hypothermia resulted in reduced hepatic damage, improvement in neurological function, normalization of glutathione levels, and selective attenuation in expression of circulating proinflammatory cytokines. These findings demonstrate that the beneficial effects of mild hypothermia in experimental AOM-induced ALF involve both antioxidant and anti-inflammatory mechanisms.     

Cryptogenic organizing pneumonia after rituximab therapy for presumed post-kidney transplant lymphoproliferative disease

Cryptogenic organizing pneumonia (COP, formerly bronchiolitis obliterans organizing pneumonia) is rare in children. We describe an 11-year-old girl with Epstein–Barr virus (EBV) reactivation/presumed post-transplant lymphoproliferative disease (PTLD) 15 months after undergoing a deceased donor kidney transplantation. Treatment with reduced immunosuppression, ganciclovir, and cytomegalovirus immunoglobulin was complicated by severe graft rejection, prompting therapy with methylprednisolone, anti-thymocyte globulin and four weekly doses of rituximab (total 1500 mg/m²). Tacrolimus- and prednisone-based anti-rejection prophylaxis was complemented with low-dose sirolimus. When the lactate dehydrogenase and uric acid levels rose 10 weeks after the first rituximab infusion and bilateral pulmonary nodules were detected by computerized tomography, recurrence of PTLD was suspected. Open lung biopsy of the clinically asymptomatic patient identified the nodules as COP, characterized by abundant CD3⁺ T-cells, few B-cells, and the absence of EBV, cytomegalovirus, or adenovirus antigens. With normalization of the peripheral B-cell count, EB viremia reappeared and persisted, despite minimal immunosuppression. Four years later, the patient was diagnosed with classical Hodgkin lymphoma-type PTLD with multiple pulmonary and abdominal nodes. This first report of rituximab-associated, pediatric COP highlights the risk of pulmonary complications after treatment with B-cell depleting agents in solid organ transplant recipients, and the importance of a histopathologic diagnosis and vigilant follow-up of such lesions.     

Vitamin D insufficiency: implications for the immune system

Chronic kidney disease (CKD) is characterized by a loss of kidney function and dysregulation of vitamin D metabolism. Well known are the defects in final activation of vitamin D to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], resulting in renal osteodystrophy. However, in recent years, 1,25(OH)₂D₃ has been identified as having effects far beyond calcium and bone metabolism. In this review, specific attention is given to the effects of 1,25(OH)₂D₃ on the immune system and the implications of vitamin D deficiency, a feature of many patients with CKD, on immune function.     

维生素D在慢性阻塞性肺疾病中发挥的效应超出其对骨骼的作用

维生素D内分泌系统调节大量基因及其相关生物过程的研究发现,让我们对维生素D与日光照射对人体健康的重要作用有了更深的认识。累积的流行病学资料显示,诸如癌症、自身免疫性疾病和慢性传染病这些高发流行性疾病都与维生素D过低有关。全世界近一半老年人及较少部分成年人的血清25-羟维生素D(25-OHD)水平低。正在进行的几项干预性研究用于分析适当的维生素D补充对慢性疾病的影响。在这项前瞻性研究中,我们主张以慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)作为补充维生素D可能对机体有益的候选疾病。流行病学研究发现血清25-OHD水平与肺功能之间存在剂量依赖性关系,因此,适当的维生素D补充并非只防治骨质疏松性骨折。根据其对免疫功能的新认识,探讨推测维生素D可下调气道的炎症免疫反应,增强抗各种微生物的先天性免疫防御功能。除了其对治疗骨质疏松的疗效,维生素D还可干预COPD的其他并存病,例如骨骼肌无力、心血管疾病及癌症。由于针对COPD的呼吸疗法不能逆转病程,因此一些探索维生素D更广的潜在价值的干预性实验成为必要。此类研究的下一步挑战是界定此类非血钙终点的最佳血清25-OHD水平。     

Genotype–Phenotype Correlations in Non-Finnish Congenital Nephrotic Syndrome

Mutations in NPHS1, which encodes nephrin, are the main causes of congenital nephrotic syndrome (CNS) in Finnish patients, whereas mutations in NPHS2, which encodes podocin, are typically responsible for childhood-onset steroid-resistant nephrotic syndrome in European populations. Genotype–phenotype correlations are not well understood in non-Finnish patients. We evaluated the clinical presentation, kidney histology, and disease progression in non-Finnish CNS cases by mutational screening in 107 families (117 cases) by sequencing the entire coding regions of NPHS1, NPHS2, PLCE1, WT1, LAMB2, PDSS2, COQ2, and NEPH1. We found that CNS describes a heterogeneous group of disorders in non-Finnish populations. We identified nephrin and podocin mutations in most families and only rarely found mutations in genes implicated in other hereditary forms of NS. In approximately 20% of cases, we could not identify the underlying genetic cause. Consistent with the major role of nephrin at the slit diaphragm, NPHS1 mutations associated with an earlier onset of disease and worse renal outcomes than NPHS2 mutations. Milder cases resulting from mutant NPHS1 had either two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. In addition, we extend the spectrum of known NPHS1 mutations by describing long NPHS1 deletions. In summary, these data demonstrate that CNS is not a distinct clinical entity in non-Finnish populations but rather a clinically and genetically heterogeneous group of disorders.Congenital nephrotic syndrome (CNS) of the Finnish type (CNF; MIM# 256300) is a recessively inherited disorder characterized by massive proteinuria at birth, a large placenta, and marked edema within the first 3 months of life.^1–4 The disease is most frequent in Finland, where its incidence is 1 per 8200 newborns.⁵ Renal histology encompasses mesangial hypercellularity and matrix expansion, progressing with age to complete mesangial sclerosis and capillary obliteration.⁶ Irregular microcystic dilation of the proximal tubules (PTD) is the most typical histologic feature, observed as early as 18 to 20 weeks of gestation^7–9 and increasing in frequency with age¹⁰; however, PTD is not observed in all cases. Ultrastructural analysis of the glomerular capillary loops shows complete foot process effacement and swelling of endothelial cells.¹¹NPHS1, encoding nephrin, was identified by positional cloning more than a decade ago and is the major gene involved in CNF in Finnish populations (98% of cases).¹² The Fin_major (c.121delCT; p.L41fs) and Fin_minor (c.3325C>T; p.R1109X) mutations account for 78 and 16% of the mutated alleles, respectively, in Finnish cases¹²; however, these mutations are rarely found in other ethnic groups.¹³ NPHS1 genetic screening in patients of non-Finnish origin has shown that the frequency of NPHS1 mutations is lower than that in Finnish patients, with such mutations accounting for 39 to 55% of cases.^14,15 Indeed, more than 140 different mutations have been identified among non-Finnish cases,^14–24 including protein-truncating nonsense mutations, frameshift small insertion/deletion mutations, and splice-site changes.^14–24 In vitro functional assays have shown that most NPHS1 missense mutations lead to retention of the protein in the endoplasmic reticulum,^25,26 resulting in a complete loss of nephrin from the cell surface. This suggests that defective intracellular nephrin trafficking, presumably as a result of protein misfolding, is a common consequence of the NPHS1 missense mutations implicated in CNS.Nephrin is a transmembrane protein of the Ig superfamily characterized by eight C2-type Ig-like domains and a fibronectin type III repeat in the extracellular region, a single transmembrane domain, and a cytosolic C-terminal end.¹² The extracellular domain of nephrin forms homodimers and heterodimers with NEPH1.^27,28 Nephrin–NEPH1 interactions control nephrin signaling,²⁹ glomerular permeability,³⁰ and podocyte cell polarity.³¹ Neph1 knockout mice present massive proteinuria within the first 2 weeks of life and renal lesions resembling CNF in humans,^32,33 suggesting that recessive inactivating mutations in the NEPH1 gene may be involved in congenital human glomerular disease; however, no mutations have yet been identified in this gene.One striking finding among patients with CNS has been the detection of mutations in the NPHS2 gene,^19,34 encoding podocin, which has been implicated mainly in early-onset steroid-resistant nephrotic syndrome (SRNS).³⁵ A recent analysis showed that as many as 51% of patients who had CNS and were of European origin had mutations in the NPHS2 gene.¹⁵ In addition to mutations in the NPHS1 and NPHS2 genes, mutations in PLCE1 and WT1, which are known to cause infantile NS, have been implicated in cases of CNS and diffuse mesangial sclerosis (DMS).^36–40 Several hereditary forms of syndromic CNS have also been described. LAMB2 mutations have been implicated in Pierson syndrome, a rare autosomal recessive disorder characterized by microcoria and other complex ocular abnormalities associated with CNS.^41,42 Moreover, patients with NS and minor structural eye defects and others with isolated NS have expanded the phenotypic spectrum of mutations in the LAMB2 gene.^43–45 Finally, mutations in the PSSD2 and COQ2 genes have been found in patients with mitochondriopathies, typically presenting primary coenzyme Q10 deficiency and profound neuromuscular symptoms and occasionally developing NS.^46–51These clinical associations confirm the genetic heterogeneity of CNS in non-Finnish patients. It remains unclear whether subtle differences in phenotype between patients who have CNS and bear NPHS1 or NPHS2 mutations could be used to guide genetic screening. Distinctive extrarenal clinical features certainly help to direct mutational screening among patients with syndromic forms of CNS; however, patients with nonsyndromic CNS may have mutations in the WT1, LAMB2, COQ2, and PDSS2 genes, making genetic screening more difficult and time-consuming. Finally, the contribution of NEPH1 mutations to CNS has never been explored. We therefore carried out a comprehensive genetic analysis in the largest multiethnic cohort of patients with CNS of non-Finnish origin reported to date, with the aim of defining the epidemiologic role of mutations in the main genes implicated in CNS and elucidating potentially novel genotype–phenotype correlations.     

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-012-9462-2) contains supplementary material, which is available to authorized users.     

A Modified Semi-Nested Multiplex Malaria PCR (SnM-PCR) for the Identification of the Five Human Plasmodium Species Occurring in Southeast Asia

We have modified an existing semi-nested multiplex polymerase chain reaction (PCR) by adding one Plasmodium knowlesi-specific nested PCR, and validated the latter against laboratory and clinical samples. This new method has the advantage of being relatively affordable in low resource settings while identifying the five human Plasmodium species with a three-step PCR.Since the first Malaysian reports and the recognition of Plasmodium knowlesi as the fifth human Plasmodium species,^1–5 there have been continuous attempts to improve the molecular detection of this Plasmodium species of simian origin.^6–11 Initially, a nested 18S small subunit (ssu) ribosomal DNA (rDNA)-based polymerase chain reaction (PCR) identified the first large cohort of naturally acquired P. knowlesi infections in humans in Borneo.¹ However, the primers used to detect the P. knowlesi DNA (Pmk8-Pmkr9) can cross-react with the Plasmodium vivax ribosomal RNA (rRNA) gene, requiring systematic confirmation by either a secondary PCR (1) or sequencing.^6,12 Since then, more specific methods using nested⁶ or non-nested PCR,^10,11 loop-mediated isothermal amplification,⁸ or real time PCR^7,9 have been developed.Plasmodium knowlesi infections in humans were reported in Vietnam¹² and this species needs to be accounted for in the newly defined national malaria elimination strategies.¹³ Applying the multiplex semi-nested (SnM) PCR published by Rubio and others¹⁴ for the detection of the four previously described human Plasmodium species, we recently showed that the malaria parasite reservoir in Central Vietnam was more complex than previously reported by standard microscopy.¹⁵ We therefore added another secondary PCR, specific for P. knowlesi, into the existing SnM-PCR to enable the detection of the five Plasmodium species within a three-step PCR.The existing SnM-PCR protocol was adapted from Rubio and others,¹⁴ and consists of a first reaction Plasmodium genus specific (Figure 1A) followed by a semi-nested PCR including the Plasmodium-specific forward (PLF) primer used in the primary PCR, plus species-specific reverse primers for Plasmodium falciparum, P. vivax, Plasmodium malaria, and Plasmodium ovale (Figure 1B). We hereby describe the newly added semi-nested (Sn-) PCR for the identification of P. knowlesi (Figure 1C). The analysis of existing oligonucleotide primers for P. knowlesi showed that the reverse complement of the PkF1140 primer designed and validated by Imwong and others⁶ was compatible with the PLF primer described by Rubio and others.¹⁴ Therefore, the Sn-PCR of P. knowlesi includes the PLF forward primer, and a species-specific reverse primer (PKR4: 3′-GAT TCA TCT ATT AAA AAT TTG CT-5′), the latter being the reverse complement of the PkF1140 primer shortened by two base pairs (bp). The specificity of the PKR4 was subsequently assessed in silico by aligning sequences for the 18S ssu rRNA genes of different human and non-human Plasmodium species (P. knowlesi, P. coatney, P. cynomolgi, P. inui, P. fragile, P. reichenowi, P. lophurae, P. gallinacae) as well as 29 P. knowlesi sequences available on GenBank (http://www.ncbi.nlm.nih.gov/genbank/index.html).Open in a separate windowFigure 1.Primers position for the modified SnM-PCR amplification are shown for the single primary reaction (A), followed by two secondary PCRs: one for the four usual human Plasmodium species (B), following protocol by Rubio and others,¹⁵ and the new secondary PCR with the PkR4 primer (C). The relative positions of the different primers used for the nested PCR amplification to detect P. knowlesi by Imwong and others⁶ are indicated below the gene: primary PCR (D) and nested PCR (E).The Sn-PCR was performed in 25 μL reaction mixture with final concentration for a 1× reaction of 1× Qiagen loading buffer (10× Qiagen buffer, Hilden, Germany), 100 μM of each dNTP (Eurogentec, Seraing, Belgium), 0.5 μM PLF, 0.2 μM PKR4, 1 Unit of Qiagen HotstarTaq plus polymerase, and 2 μL DNA (a 1/500 dilution of the primary PCR product was used as the template). Cycling conditions were as follows: a 5 min denaturation and activation of the Qiagen HotstarTaq plus polymerase step at 94°C, followed by 30 cycles of 20 sec at 51°C for annealing, followed by elongation at 72°C for 1 min, and a 30 sec denaturation at 94°C. The final cycle was followed by an extension time of 10 min at 72°C. The PCR products were detected by 2% agarose gel stained with ethidium bromide, and visualized under UV light.Reference blood samples from P. knowlesi-infected patients (N = 13) were kindly provided by the IMR, Kuala Lumpur. Furthermore, 80 blood samples (filter paper) from malaria-infected patients collected during a previous survey carried out in Vietnam¹⁵ were used to further validate the specificity of the PLF-PKR4 primers. The DNA extraction was done using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany).Primary and nested PCR products were cloned into plasmid vectors (pCR4-TOPO vector) and transformed into Mach1 T1^B Escherichia coli cells using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Thirty bacterial colonies, 10 from two distinct P. knowlesi samples harboring the amplified primary PCR fragment, and 10 harboring the secondary PCR fragment (five colonies for each of the two P. knowlesi samples) were purified and sequenced. In addition, DNA extracts from the 13 P. knowlesi reference samples were subjected to the new Sn-PCR and the amplified PCR fragments of expected size (actual size amplicon = 498 bp), were cut out of the agarose gel, purified, cloned, and sequenced. The sequences obtained were analyzed with the BioEdit program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and compared with available sequences in GenBank. All sequences obtained from plasmids with the primary and nested PCR inserts were confirmed as P. knowlesi.The sensitivity and specificity of our Sn-PCR protocol was assessed using two previously published nested PCR protocols for the detection of P. knowlesi: one using Pmk8-Pmkr9 primers¹ (hereafter called PCR1) and the one using the PkF1150-{"type":"entrez-protein","attrs":{"text":"PkR15560","term_id":"1314896832","term_text":"PKR15560"}}PkR15560 primers⁶ (hereafter called PCR2). The relative sensitivity was determined by a 10-fold serial dilution of the P. knowlesi H strain (Plasmodium knowlesi Genomic DNA-P. knowlesi H strain, MRA-456G obtained at the MR4 [http://www.mr4.org/]) using DNA extract from human blood collected from healthy donors. The analytic sensitivity was determined using a 10-fold serial dilution of the Primary PCR product of PCR 1 and 2 (using primers rPLU1 and 5). This PCR product was first cloned, extracted, and sequenced. The extracted plasmid DNA concentration was determined using three measurements of the Nanodrop (Thermo Scientific, Landsmeer, The Netherlands). A 10-fold serial dilution of the plasmid DNA containing the P. knowlesi fragment was made to determine the sensitivity of the different PCRs. The PCR1 showed the highest in both experiments with an analytical sensitivity at dilutions as low as 1 fg/μL compared with the 100 fg/μL for PCR2 and our Sn-PCR. In both panels the P. knowlesi MR4 H strain as well as the plasmid dilution series, our Sn-PCR and the PCR2 showed the same sensitivity.The specificity of the three protocols was tested using different genomic DNA controls from Plasmodium species originating from monkeys (1 P. cynomolgy, 1 P. simium, 1 P. fragile; reference strains from NIMPE, Hanoi), and humans (4 P. vivax, 2 P. falciparum, 1 P. ovale, and 1 P. malariae; reference strains from ITM, Antwerp), 1 P. knowlesi (IMR, Kuala Lumpur) as well as genomic DNA (gDNA) of non-Plasmodium origin (1 Leishmania donovani, 1 Schistosoma mansoni, 1 Trypanosoma cruzi, 1 HIV provirus, 1 Mycobacterium tuberculosis, and 1 Mycobacterium ulcerans). Our Sn-PCR and PCR2 showed no cross-reaction, whereas PCR1 showed a false positive P. knowlesi with a 153 bp band for one of the four P. vivax controls.The specificity was further tested with the 80 clinical samples, previously analyzed by SnM-PCR for the identification of the four human Plasmodium species.¹⁵ Filter paper dried blood spots were then tested for the presence of P. knowlesi using the three nested PCR protocols (

Distribution,evolution, and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA clusters

Most of our understanding of Drosophila heterochromatin structure and evolution has come from the annotation of heterochromatin from the isogenic y; cn bw sp strain. However, almost nothing is known about the heterochromatin’s structural dynamics and evolution. Here, we focus on a 180-kb heterochromatic locus producing Piwi-interacting RNAs (piRNA cluster), the flamenco (flam) locus, known to be responsible for the control of at least three transposable elements (TEs). We report its detailed structure in three different Drosophila lines chosen according to their capacity to repress or not to repress the expression of two retrotransposons named ZAM and Idefix, and we show that they display high structural diversity. Numerous rearrangements due to homologous and nonhomologous recombination, deletions and segmental duplications, and loss and gain of TEs are diverse sources of active genomic variation at this locus. Notably, we evidence a correlation between the presence of ZAM and Idefix in this piRNA cluster and their silencing. They are absent from flam in the strain where they are derepressed. We show that, unexpectedly, more than half of the flam locus results from recent TE insertions and that most of the elements concerned are prone to horizontal transfer between species of the melanogaster subgroup. We build a model showing how such high and constant dynamics of a piRNA master locus open the way to continual emergence of new patterns of piRNA biogenesis leading to changes in the level of transposition control.Over the course of evolution, transposable elements (TEs) have accumulated in the genomes of eukaryotes, where they can account for up to 85% of the DNA (1). Most of these sequences have lost their ability to transpose. They are now stable components of the genomes. Their conservation throughout evolution suggests that they may confer advantageous effects to their hosts. However, transposition of the copies that remain functional could generate deleterious mutations if they were not severely repressed by their host. RNAi, which is a gene-silencing mechanism triggered by small RNAs (reviewed in ref. 2), has been identified as being the main cellular machinery involved in the “taming” of TEs (reviewed in refs. 3–5). RNAi pathways involve small RNAs of diverse families. Among them, Piwi-interacting RNAs (piRNAs) have been shown to be involved in TE silencing in the Drosophila ovary. These piRNAs, 23–29 nt long, are bound by the Argonaute proteins Piwi, Argonaute 3, or Aubergine. They are produced by discrete genomic loci named piRNA clusters, which have been described as containing vestiges of TEs (6). One of these loci, the flamenco (flam) locus, extends over 180 kilobases (kb) on the Drosophila X chromosome. It is proximal to the DISCO interacting protein 1 gene (DIP1) and close to pericentromeric heterochromatin. Before the identification of piRNAs, this locus had been shown to regulate the Gypsy retrotransposon (7, 8). Desset et al. (9) identified a locus called COM responsible for the control of ZAM and Idefix retrotransposons. Later, flam and COM were shown to be one and the same large master locus for regulation of at least these three retrotransposons: ZAM, Idefix, and Gypsy (reviewed in ref. 10). Control of different retrotransposons by the flam locus had been shown for diverse flam alleles: for ZAM and Idefix in the Rev line (9) and for Gypsy and ZAM in flam mutant lines (11). Brennecke et al. (6) showed the potential for the flam cluster to produce 79% of all ovarian piRNAs that target ZAM and 30% and 33% of those matching Idefix and Gypsy, respectively. It was further reported that a functional pathway links flam piRNA to Gypsy suppression. Indeed, a substantial reduction of piRNA homologous to Gypsy was observed in flam mutant lines that are permissive to Gypsy transposition (12). Still, the molecular rules allowing this locus to control several retrotransposons are far from being understood.In previous work, we isolated Drosophila lines in which ZAM and Idefix are either silenced (i.e., “stable”) or derepressed (i.e., “unstable”). Over the course of a P-element mediated mutagenesis performed on a stable line called w^IR6, we isolated a line, Rev, derived from w^IR6, in which ZAM and Idefix are both derepressed (13–15). Novel mobilizations are regularly observed, and reporter genes containing ZAM or Idefix fragments are expressed in the Rev line, although silenced in w^IR6. We further reported that the observed release in ZAM and Idefix silencing was due to an unidentified mutation having affected the flam locus (9).Bergman et al. (16) laid out the hypothesis that β-heterochromatic TE nests will act as transposon traps for new TE invasions by horizontal transfer providing an “adaptive immunity” to the host genome. Here, we wondered whether specifically piRNA loci like flam might act as a trap for TEs. If so, any trapped TE that inserted into a piRNA cluster should immediately be silenced and further transposition events should not occur. Then, a piRNA locus would be predicted to contain only one copy of a TE per family. To test this model, we first revisit the annotation of the flam locus. We show that, as predicted, most of the TEs in the flam locus are present only once. More than half of the flam locus is made up of recent TEs, and two-thirds of them arose from horizontal transfer, confirming the trap model. We then evaluate the structure of the flam region in permissive and repressive strains. Our data indicate that strains permissive for the mobility of ZAM and Idefix display large deletions that removed ZAM and Idefix sequences present in flam. These results reveal a tight correlation between the presence of these retrotransposons in this locus and their silencing. They also highlight how transposition bursts may occur due to the high molecular dynamics of piRNA clusters like flam.