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Reproductive fitness of the human Y

Repping, S.

Publication date 2003

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Citation for published version (APA): Repping, S. (2003). Reproductive fitness of the human .

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Download date:23 Sep 2021 Chapter Chapter

Thee use of spermHALO-FISH to determine DAZDAZ copy number

SjoerdSjoerd Repping, Jan W.A. de Vries, Saskia K.M. van Daalen, Cindyy M. Korver, Nico J. Leschot and Fulco van der Veen

MolecularMolecular Human Reproduction 9:183-188, 2003 ChapterChapter 2

Abstract t

Thee AZFc region of the human Y chromosome is frequently deleted in men with spermatogenicc failure and contains many multicopy . The best-characterized gene familyy within this region is the Deleted in Azoospermia (DAZ) gene family, which is presentt in four nearly identical copies. Recent reports claim deletions of some but not all DAZDAZ genes. The assays used in these studies, however, are unable to provide conclusive evidencee on the number of DAZ genes. Inn this study we show that with the use of highly decondensed sperm nuclei with largee DNA domains (spermHALO) it is possible to determine the number of DAZ genes accurately.. Using this Fluorescent in-situ hybridization (FISH) technique, which has both highh resolution and high range, we show that in 10 normospermic men, in which PCR digestt assays indicated a deletion of one or more DAZ genes, all four DAZ genes were present.. Also we confirmed previous findings of a deletion of two DAZ genes in two men andd identified a man with six DAZ genes. Ourr results indicate that spermHALO-FISH allows an accurate determination of DAZDAZ gene copy number, while PCR digest assays do not. Therefore, confirmation of positivee results from PCR digest assays with spermHALO-FISH is essential. Furthermore, thee spermHALO-FISH technique should prove useful as a genetic mapping technique in otherr regions of the Y chromosome and similar repetitive regions throughout the genome. .

22 2 SpermHALO-FISHH and DAZ copy number

Introduction n

Deletionn of Y-chromosomal DNA is the most common molecular cause of spermatogenicc failure in men. Four common classes of such deletions have been described:: AZFa, P5/proximal PI (AZFb), P5/distal PI, and AZFc deletions (Vogt et al., 1996;; Repping et al., 2002). Deletions of the AZFc region are found in ~10% of men withh azoospermia (Reijo et al., 1996; Kremer et al., 1997; Kuroda-Kawaguchi et al., 2001).. The AZFc region contains seven gene families with a total of 18 gene copies that aree all expressed exclusively or predominantly in the testis (Kuroda-Kawaguchi et al,, 2001).. One of these genes is the Deleted in Azoospermia {DAZ) gene. Although the precisee function of DAZ is unclear, studies in several species indicate that disruption of thiss gene leads to spermatogenic failure (Eberhart et al., 1996; Ruggiu et al., 1997; Houstonn and King, 2000).

DAZDAZ was originally thought to be single copy, but the presence of slight differencess in sequenced cosmids from a single man indicated that there were at least twoo copies (Reijo et al., 1995; Saxena et al., 1996). Subsequently, several studies claimedd that there were anywhere between three and seven copies of the gene (Glaser ett al., 1997; 1998; Yen et al., 1997; Yen, 1998). With the use of conventional Fluorescencee in-situ hybridization (FISH) techniques and the complete nucleotide sequencee of the AZFc region, we now know that in fact there are four DAZ genes on the humann Y chromosome, arranged in two clusters with two genes in head-to-head orientationn (Saxena et al., 2000; Kuroda-Kawaguchi et al., 2001).

Recently,, two studies claim to have found deletions of some but not all copies of thee DAZ genes in subfertile men, suggesting such deletions as causes of spermatogenic failuree (de Vries et al., 2002a; Fernandes et al,, 2002). Consequently, the search for variationn in DAZ gene copy number and the role of such variation in spermatogenic failuree is ongoing. However, determining the precise number of DAZ genes and searchingg for possible deletions of some but not all gene copies is difficult. The reason is thatt members of gene families on the human Y chromosome, such as DAZ, show relativelyy few differences in nucleotide sequence thereby precluding the use of PCR assayss to allow distinction of the different copies.

Too elucidate the DAZ gene copy number and to search for possible deletions of somee copies of DAZ, two methods are available: PCR digest assays, and FISH techniques.. The PCR digest assays make use of the subtle nucleotide variations in differentt copies of DAZ, so-called sequence family variants (SFV) or single nucleotide variantss (SNV). However, a drawback of these PCR digest assays is that the variants studiedd appear to be polymorphic, making interpretation of the results difficult (de Vries ett al., 2002a). FISH, on the other hand, is a powerful tool in highly repetitive regions

23 3 ChapterChapter 2

suchh as AZFc. By using DAZ-specific cosmids as probes and leukocyte interphase nuclei ass targets (IP-FISH), it is possible to determine the number of DAZ gene clusters. Fibre- FISH,, which makes use of extended chromatin fibres from leukocytes, allows the determinationn of the number of genes within a cluster. Although useful, a drawback of thesee two techniques is that it is impossible to visualize directly both clusters with all fourr genes in a single microscopic image. Consequently, a deletion of a single DAZ gene copyy cannot be excluded with these techniques. Heree we aim to provide conclusive evidence on DAZ gene copy number by using spermHALO-highlyy decondensed sperm nuclei with large DNA loop domains-as a templatee for FISH. Furthermore, we compare the results from spermHALO-FISH with resultss from conventional FISH techniques and PCR digest assays.

Materialss and methods

PatientPatient samples Inn total we investigated 22 fertile semen donors who participated in our programmee for artificial insemination with donor semen and six subfertile patients who weree previously identified as having a reduced DAZ gene copy number by SFV and conventionall FISH techniques (de Vries et al., 2002a). From each man, blood was drawnn for isolation of DNA and isolation of leukocytes. A semen sample was obtained andd used for spermHALO preparations as described below. Thiss study was approved by the Institutional Review Board of the Academic Medicall Center and all donors and patients gave informed consent.

PCRPCR digest assays Analysiss of SFV was performed using markers sY581, sY586 and sY587 as describedd previously (see Table 1) (Saxena et al., 2000; de Vries et al., 2002a), Analysiss of SNV and of DAZ-RRM3 and Y-DAZ3 was performed according to Fernandes ett al. (2002) (see Table 1).

24 4 SpermHALO-FISHH and DAZ copy number

Tablee 1. Overview of PCR digest assays. Variantss Location SNVV I G G DAZ4 DAZ4 A A DAZl, DAZl,DAZ2,DAZ2, DAZ3

SNVII I G G DAZl DAZl A A DAZ2, DAZ2,DAZ3,DAZ3, DAZ4

SNVV III ( =SY586 ) ) T T DAZ2 DAZ2 C C DAZl, DAZl,DAZ3,DAZ3, DAZ4

SNVV IV T T DAZ2 DAZ2 C C DAZl, DAZl,DAZ3,DAZ3, DAZ4 SNVV V(=sY587) C C DAZ3, DAZ3,DAZ4 DAZ4 T T DAZl, DAZl,DAZ2 DAZ2

SNVV VI G G DAZ4 DAZ4 T T DAZl, DAZl,DAZ2,DAZ2, DAZ3 sY581 1 C C DAZl, DAZl,DAZ4 DAZ4 T T DAZ2, DAZ2,DAZ3 DAZ3 DAZ-DAZ- RRM 3 + + DAZl, DAZl,DAZ4 DAZ4 DAZ2, DAZ2,DAZ3 DAZ3 Y-DAZ3 Y-DAZ3 + + DAZ3 DAZ3 DAZl, DAZl,DAZ2,DAZ2, DAZ4 Note:: SNV = Single Nucleotide Variant

SpermHALOSpermHALO preparations Too overcome the limitations of IP-FISH and fibre-FISH, we applied a different methodd using highly decondensed sperm (spermHALO) as a template for FISH. By exposingg sperm to dithiothreitol (DTT), a gradual swelling of the nucleus is achieved withh chromatin fibres extending from the sperm head while remaining attached to the nuclearr annulus (see Figure 1) (Ward et al., 1989). These DNA domain loops consist of linearr DNA and therefore provide optimal resolution for FISH analysis. Furthermore, one cann readily observe the entire cell in a single microscopic image without confounding effectss of neighbouring cells (Figure 2D). SpermHALOO were prepared from semen samples that were obtained by masturbationn after >2 days of sexual abstinence and subsequently cryopreserved (for thee subfertile men we used fresh ejaculate instead of frozen samples). After thawing, spermm were separated from seminal plasma by washing twice with 10 mmol/l Tris-HCI

(100 minf 300 g). Subsequently, the pellet was resuspended in 5 ml 0.75% sodium dodecyll sulphate (SDS), 10 mmol/l Tris-HCI, mixed, incubated for 5 min at room temperature,, and mixed again. The SDS solution was removed after centrifugation (10 min,, 300 g) and the pellet was washed with 10 mmol/l Tris-HCI (10 min, 300 g). The pellett was then diluted in 10 mmol/l Tris-HCI to a concentration of 25xl06 sperm per ml. Thee sample was diluted 1:9 in extraction buffer (2 mol/i NaCI, 10 mmol/l DTT, 10 mmol/ll Tris, pH 7.4) and incubated for 0.5 to 4 min at C (the duration of incubation

25 5 ChapterChapter 2 variedd for different patients as the response to the extraction buffer was variable; also, thee concentration of DTT was adjusted for some semen samples because of under- or overswellingg of the sperm nuclei in these samples). After extraction, small droplets of thee samples were dropped on ice-cold slides and incubated at C for 25 min. Subsequently,, the extraction buffer was removed by gently dropping 10 mmol/l Tris-HCI ontoo the slides. Finally, slides were air-dried and dehydrated using a 70, 96 and 100% ethanoll series. Slides were used immediately for FISH analysis.

Figuree 1. Gradual swelling of a sperm nucleuss resulting in spermHALO (highly decondensedd sperm nuclei with large DNAA domains) formation. A, Non- swollenn sperm nucleus. B, Slightly swollenn sperm nucleus. C, Swollen spermm nucleus with small HALO-like structures.. D, Fully swollen sperm nucleuss with large DNA-HALO. The DNA fibress remain attached to the nuclear annuluss (dark stain top right) and form aa HALO-like structure. This spermHALO cann be observed in a single microscopic imagee (xlOO magnification) without confoundingg effects of neighbouring spermm cells.

FISH FISH

Onee or two-colour FISH was performed as described previously (Saxena et al.,

2000;; de Vries et al., 2002a). Slides with either interphase nuclei (IP-FISH), extended chromatinn fibres (fibre-FISH) or highly decondensed sperm nuclei with large DNA domainss (spermHALO-FISH) were used as hybridization targets. Two sequenced DAZ cosmidss were used as probes: cosmid 18E8 (encompassing both 5' ends and the interveningg sequence of two neighbouring DAZ genes) and cosmid 46A6 (encompassing thee 3' end and 35 kb of flanking sequence). To determine the number of DAZ clusters withh the use of IP-FISH, the number of signals for probe 18E8 was counted in >200 nuclei.. For fibre-FISH and spermHALO-FISH, the number of DAZ genes was determined byy examining the order of the signals (which appear as beads-on-a-string) after two- colourr hybridization with both DAZ probes.

26 6 SpermHALO-FISHH and DAZ copy number

AccessionAccession numbers GenBankk accession numbers (http://www.ncbi.nlm.nih.gov/Genbank) are as follows.. SY581: G63906; SY586 = SNV III: G63907; sY587 = SNV V: G63908; SNV I: G73167;; SNV II: G73163; SNV IV: G73168; SNV VI: G73169; Y-DAZ3: G73170; DAZ- RRM3:: G73171. Online Mendelian Inheritance in Man (OMIM) (http://www.ncbi.nlm.nih.-gov/Omim/):: for AZFc: MIM 40024.

Results s

PCRPCR digest assays Analysiss of PCR digest assays in the 22 fertile semen donors gave a diversity of results.. Interpretation of these results suggested a deletion of at least one DAZ gene in 10/222 men examined. One man appeared to have a deletion of DAZ3 only, eight men showedd PCR digest results suggesting a deletion of DAZ2 and DAZ4 and one man appearedd to have retained only DAZ1 (Table 2). All six patients who were previously identifiedd as having a reduced DAZ gene copy number by SFV and conventional FISH techniques,, showed aberrations in SNV as well. More precisely, the results indicated a deletionn of two genes in one man, a deletion of three genes in four men and in one man PCRR digest results could be interpreted as the absence of all four DAZ genes (Table 3).

ConventionalConventional FISH techniques Inn contrast to the large number of aberrations found with PCR digest assays, IP- FISHH using probe 18E8 showed two signals, representing two DAZ gene clusters, in all butt donor ID20 (see Figure 2A). In this donor, who appeared to be deleted for DAZ2 andd DAZ4 by SNV analysis, IP-FISH showed three signals in the majority of nuclei examinedd indicating the presence of three DAZ gene clusters (Figure 2B, C). The presencee of three DAZ gene clusters in this donor had been noted before in a study that determinedd the number of DAZ gene clusters in sperm nuclei (de Vries et al., 2002b). IP-FISHH in the six patients was as reported previously, i.e. all patients had only one DAZ clusterr (de Vries et al., 2002a). Fibre-FISH analysis using extended chromatin fibres fromm leukocytes showed the presence of two DAZ genes in head-to-head orientation in alll donors, including the donor with three clusters (Figure 2d). Again, fibre-FISH in the sixx patients was as reported previously, i.e. all patients had two DAZ genes in head-to- headd orientation (de Vries et al., 2002a).

27 7 ChapterChapter 2

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28 8 SpermHALO-FISHH and DAZ copy number

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HH H 133£ 1g3 H H Si i h-h- h- 4->> QQJ . .QJJ CO Figuree 2. FISH results using interphase nuclei HH U U U (- U andd extended DNA fibres as a template. A, I' ' Interphasee nucleus probed with DAZ probe 18E8 üü £ showingg two signals representing two DAZ gene cc c clusters.. B, Interphase nucleus from donor ID20 Inn 8 [JJ u u u {^ u QJ J showingg three DAZ gene clusters. C, Bar graph "O O showingg the results from spot counting in donor ^^ > ID200 and in a control man with two DAZ gene ü>> g> clusters.. D, Extended DNA fibre probed with UU U U U H 22 £ cosmidd 18E8 (red) and cosmid 46A6 (green) showingg two DAZ genes with head-to-head Eo o orientation.. Probe 18E8 detects the 5' end of QJJ E DAZDAZ and the sequence between the two DAZ (133 01 33 < < < g Ö ££ CL genes,, while probe 46A6 detects the 3' end of TJJ m cc i_ DAZDAZ and flanking sequence. —— O QJJ *- ^-- QJ 1<<33 U< 1<3 << < .22 rö JUU (O roro ro ITJ fo -OO .2 ** i oo o o o o o co UU

29 9 ChapterChapter 2

SpermHALO-FISH SpermHALO-FISH Wee were able to determine the DAZ gene copy number in all donors and in both patientss for whom sperm were available (from four patients no sperm were available; seee Table 3). In 21 donors we clearly observed four DAZ genes arranged in two clusters withh two genes in head-to-head orientation (Table 2, Figure 3A). Similarly, we observed onlyy a single DAZ cluster with two genes in both patients previously identified as having aa reduced DAZ gene copy number (Table 2, Figure 3B). Finally, spermHALO from donor ID20,, in which IP-FISH showed three DAZ clusters, showed the presence of six DAZ geness organized in three identical clusters with two genes in head-to-head orientation (Figuree 3C).

Discussion n

Inn this study we showed that spermHALO-FISH allows the precise determination off DAZ gene copy number. In contrast, PCR digest assays gave false positive results in 100 men. In nine of these men spermHALO-FISH clearly showed the presence of four DAZDAZ copies, and in one man a total of six DAZ genes were found. We also showed that alll but one sperm donor carried four DAZ copies and confirmed previous findings in two patientss who were deleted for two DAZ genes. Alll four common classes of Y-chromosome deletions that have been described to datee result in the deletion of all copies of at least two gene families (Blanco et al., 2000; Kampp et al., 2000; Sun et al., 2000; Kuroda-Kawaguchi et al., 2001; Repping et al., 2002).. However, not much is known about deletions involving only some of the genes of aa Y-chromosome gene family. Recently, two reports have described the deletion of somee of the four DAZ genes in a group of subfertile men (de Vries et al., 2002a; Fernandess et al., 2002). Our results from spermHALO-FISH here further confirm this finding.. The main drawback in searching for such deletions is the lack of an appropriate screeningg technique. Investigators have tried to use subtle nucleotide differences betweenn different gene copies of the same gene family to search for such deletions. Althoughh relatively fast, these techniques often result in false-positive findings as shown inn this study. While all men with a reduced DAZ gene copy number were detected with PCRR digest assays (100% sensitivity), the specificity of PCR digest assays was only 38% (onlyy 6/16 men with PCR digest assay aberrations had indeed a deletion of two DAZ genes).. Also, in four of the six men with only two DAZ genes, PCR digest assays indicatedd that there was only one DAZ gene present. One man (AMC0135) had aberrationss in eight of the nine PCR digest assays that when interpreted together could suggestt the absence of all four DAZ genes; again, spermHALO-FISH showed the presencee of two DAZ genes.

30 0 SpermHALO-FISHH and DAZ copy number

Figuree 3. FISH on spermHALO (highly decondensed sperm nuclei with large DNA domains) showingg two, four and six DAZ genes. All spermHALO were probed with DAZ probes 18E8 (red) andd 46A6 (green). DAZ gene clusters can be seen as a thread of signals going from green to red too green. A, SpermHALO from a normospermic donor (ID07) showing two DAZ gene clusters with aa total of four DAZ genes. PCR digest assays indicated a deletion of DAZ2 and DAZ4 in this man. B,, SpermHALO from a subfertile man (AMC0135) showing one DAZ cluster with two DAZ genes. PCRR digest assays indicated a deletion of DAZ1, DAZ2, DAZ3 and DAZ4 in this man. C, SpermHALOO from donor ID20. Although PCR digest assays indicated a deletion of DAZ2 and DAZA inn this man, this spermHALO clearly shows three DAZ clusters and a total of six DAZ genes.

FISHH techniques are apparently far more reliable than PCR digest assays, but requiree expertise and are laborious. IP-FISH allows the determination of the number of DAZDAZ gene clusters, while fibre-FISH can subsequently determine the number of genes withinn such a cluster. These techniques, however, cannot rule out the deletion of a singlee DAZ gene. On the other hand, the technique of spermHALO-FISH as described here,, proved to be highly useful in determining the precise DAZ gene copy number. This techniquee has previously been used to examine the structure and cellular function of the

31 1 ChapterChapter 2

spermm nuclear matrix, but not as a genetic mapping technique (De Lara et al., 1993; Choudharyy et al., 1995; Nadel et al., 1995; Yaron et al., 1998; Schmid et al., 2001). Thee highly decondensed DNA loop domains provide both high resolution and high range (severall Mb), thereby forming an ideal template for FISH. Furthermore, due to the attachmentt of the DNA loop domains to the nuclear annulus, one can easily identify the totall amount of DNA originating from a single cell, thereby eliminating confounding effectss from neighbouring cells. Thee fact that PCR digest assays are not highly reliable in determining DAZ gene copyy number indicates that the nucleotide differences on which these techniques rely aree polymorphic. The absence of one of the two variants apparently does not indicate thee absence of the gene that contains the variant but more likely means that both geness contain the same variant. Recent reports indicate that Y-Y gene conversion probablyy underlies the polymorphic nature of these variants (S. Rozen et al., 2003; H. Skaletskyy et al., 2003). Gene conversion occurs when homologous recombination betweenn repetitive structures, which are abundant on the human Y chromosome, gives risee to unidirectional copying (Figure 4). In the absence of meiotic recombination with a sisterr chromosome, this process seems to be highly frequent on the Y chromosome (S. Rozenn et al., 2003). Our results here show that the degree of variation differs between differentt nucleotide variants, ranging from zero (markers SNV-II, sY581, sY587 and DAZ-RRM3)) to nine (marker SNV-VI) out of 22 samples examined (Table 2). This variationn could indicate that some sites are more susceptible to gene conversion events thann others or that some of these variants have occurred quite recently in evolution.

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Figuree 4. Gene conversion. Unidirectional gene-copying can occur through homologous recombinationn between two DAZ genes (which are >99% identical) leading to absence of a single nucleotidee variant in the next generation (examples 2 and 3). The absence of such a variant can bee mistaken as a deletion as shown in the gel image (top right).

32 2 SpermHALO-FISHH and DAZ copy number

Ass a result of these gene conversion events, it is easy to misinterpret the absencee of a subtle nucleotide variant as a deletion event. Therefore, when finding aberrationss with assays that rely on these polymorphic differences, spermHALO-FISH (or,, when not possible, conventional IP-FISH and fibre-FISH) should be performed to confirmm or contradict these findings. The technique of spermHALO-FISH is highly reliable inn determining DAZ gene copy number and provides a useful tool in searching for deletionss of some but not all of the DAZ genes in subfertile men. Besides its use for determiningg DAZ gene copy number, it should also be useful in determining gene copy numberr of other multicopy genes on the Y chromosome. Furthermore, its high resolutionn and high range make it a potentially useful method for clarifying the genomic structuree in other repetitive regions throughout the genome.

Acknowledgements s Wee thank Laura Brown and David C. Page for the use of cosmids 18E8 and 46A6.

References s Blanco,, P., Shlumukova, M., Sargent, C. A., Jobling, M., Affara, N., and Hurles, M. E. (2000) Divergentt outcomes of intrachromosomal recombination on the human Y chromosome: male infertilityy and recurrent polymorphism. J.Med.Genet, 37, 752-758. Choudhary,, S. K., Wykes, S. M., Kramer, J. A., Mohamed, A. N,, Koppttch, F., Nelson, J. E., and Krawetz,, S. A. (1995) A haploid expressed gene cluster exists as a single chromatin domain in humann sperm. J.Biot.Chem., 270, 8755-8762. Dee Lara, J., Wydner, K. L., Hyland, K. M., and Ward, W. S. (1993) Fluorescent in situ hybridizationn of the telomere repeat sequence in hamster sperm nuclear structures. J.Cell Biochem.,Biochem., 53, 213-221. dee Vries, J. W., Hoffer, M. J. V., Repping, S., Hoovers, J. M. N., Leschot, N. J., and van der Veen, F.. (2002a) Reduced copy number of the DAZ genes in sub- and infertile men. Fertil.Steril., 77, 68-75. . dee Vries, J. W,, Repping, S., van Daalen, S. K., Korver, C. M., Leschot, N. J., and van der Veen, F. (2002b)) Clinical relevance of partial AZFc deletions. Fertil.Steril., 78, 1209-1214. Eberhart,, C. G., Maines, J. Z., and Wasserman, S. A. (1996) Meiotic cell cycle requirement for a flyy homologue of human Deleted in Azoospermia. Nature, 381, 783-785. Fernandes,, S., Huellen, «., Goncalves, J., Dukal, H., Zeisler, J., Rajpert, D. M., Skakkebaek, N. E., Habermann,, B., Krause, W., Sousa, M. et at. (2002) High frequency of DAZ1/DAZ2 gene deletions inn patients with severe oligozoospermia. Mol.Hum.Reprod., 8, 286-298. Glaser,, B., Hierl, T., Taylor, K., Schiebel, K., Zeitler, S., Papadopoullos, K., Rappold, G., and Schempp,, W. (1997) High-resolution fluorescence in situ hybridization of human Y-linked genes onn released chromatin. Chromosome Res., 5, 23-30. Glaser,, B., Yen, P. H., and Schempp, W. (1998) Fibre-fluorescence in situ hybridization unravels apparentlyy seven DAZ genes or pseudogenes clustered within a Y-chromosome region frequently deletedd in azoospermia males. Chromosome Res., 6, 481-486. Houston,, D. W. and King, M. L. (2000) A critical role for XDAZI, a germ plasm-localized RNA, in thee differentiation of primordial germ cells in Xenopus. Development, 127, 447-456. Kamp,, C, Hirschmann, P., Voss, H., Huellen, K., and Vogt, P. H. (2000) Two long homologous retrovirall sequence blocks in proximal Yqll cause AZFa microdeletions as a result of intrachromosomall recombination events. Hum.Mol.Genet., 9, 2563-2572.

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