Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on May 4, 2007
Human Molecular Genetics 2007 16(13):1587-1592; doi:10.1093/hmg/ddm108

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L1 retrotransposition can occur early in human embryonic development

José A.J.M. van den Hurk¹, Iwan C. Meij², Maria del Carmen Seleme³, Hiroki Kano³, Konstantinos Nikopoulos^1,8, Lies H. Hoefsloot¹, Erik A. Sistermans¹, Ilse J. de Wijs¹, Arijit Mukhopadhyay^1,8, Astrid S. Plomp^4,5, Paulus T.V.M. de Jong^4,6,7, Haig H. Kazazian³ and Frans P.M. Cremers^1,8,*

¹ Department of Human Genetics, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands, ² Max Delbrück Centre for Molecular Medicine, 13125 Berlin, Germany, ³ Department of Genetics, University of Pennsylvania School of Medicine, Pennsylvania, PA 19104-6145, USA, ⁴ Department of Neuromedical Genetics, The Netherlands Institute for Neuroscience, 1105 BA Amsterdam, The Netherlands, ⁵ Department of Clinical Genetics, ⁶ Department of Ophthalmology, Academic Medical Centre, 1105 AZ Amsterdam, The Netherlands, ⁷ Department of Epidemiology and Biostatistics, Erasmus Medical Centre, 3015 GD Rotterdam, The Netherlands and ⁸ Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6525 GA Nijmegen, The Netherlands

^* To whom correspondence should be addressed at: Department of Human Genetics 855, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: +31 243613750; Fax: +31 243668752; Email: f.cremers{at}antrg.umcn.nl

Received April 13, 2007; Accepted April 19, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
L1 elements are autonomous retrotransposons that can cause hereditary diseases. We have previously identified a full-length L1 insertion in the CHM (choroideremia) gene of a patient with choroideremia, an X-linked progressive eye disease. Because this L1 element, designated L1_CHM, contains two 3'-transductions, we were able to delineate a retrotransposition path in which a precursor L1 on chromosome 10p15 or 18p11 retrotransposed to chromosome 6p21 and subsequently to the CHM gene on chromosome Xq21. A cell culture retrotransposition assay showed that L1_CHM is one of the most active L1 elements in the human genome. Most importantly, analysis of genomic DNA from the CHM patient's relatives indicated somatic and germ-line mosaicism for the L1 insertion in his mother. These findings provide evidence that L1 retrotransposition can occur very early in human embryonic development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
LINE-1 (long interspersed nuclear elements-1) or L1 elements are the most abundant autonomous retrotransposons in the human genome, accounting for 17% of its mass. Although the vast majority of L1s are functionally inactive, 80 to 100 L1 elements remain capable of retrotransposition in the average human genome (1). Most of these active L1s belong to the transcribed group a (Ta) subset and are polymorphic with respect to their presence or absence and retrotransposition capability (2,3). A functional L1 element measures 6 kb and consists of a 5'-untranslated region (UTR) with internal promoter activity, two open-reading frames (ORFs) and a 3'-UTR with a polyadenylation signal followed immediately by a poly(A) tail (4). Retrotransposition of L1 elements involves transcription, reverse transcription and integration into a new genomic location. Reverse transcription and integration occur in a coupled reaction termed target-primed reverse transcription (5). L1 retrotransposons have altered the human genome through a variety of mechanisms other than their own mobilization (reviewed in 6). L1 elements can transport downstream flanking DNA to a new genomic location in a process called 3'-transduction. This occurs when the L1 polyadenylation signal is bypassed in favor of a downstream polyadenylation site (7–9). L1s are potential generators of genomic deletions (10,11).

At present, information about either the timing or the cell-type specificity of human L1 retrotransposition events is limited. In vitro studies have demonstrated that a variety of human transformed or immortalized cultured cells can accommodate retrotransposition (12–14). In addition, a recent report documented retrotransposition of a human L1 element both in cultured rat neural progenitor cells and in the brain of transgenic mice (15). Also, an L1 insertion disrupting the adenomatous polyposis coli (APC) gene has been identified in a colorectal tumor, but was absent in the surrounding constitutional tissue of the patient (16). It thus seems that L1 retrotransposons can mobilize in some somatic cells. However, L1 elements can only propagate and expand if retrotranspositions occur in primordial germ cells, their derivatives in the germ line or early in embryonic development. A mouse model for human L1 retrotransposition (17) and the investigation of a disease-producing L1 insertion into the cytochrome b(–245) beta subunit gene (18) have indicated that L1 retrotransposition can occur during meiosis I in both male and female germ cells. An insertion of an engineered human L1 element at an early stage in development of a transgenic mouse has also been reported (19). It should be noted, however, that transcription of this L1 transgene was under the control of the mouse RNA polymerase II large subunit promoter in addition to its endogenous promoter.

L1 retrotransposons can cause hereditary diseases by integrating into genes (20,21 and references therein). Previously, we identified an L1 insertion in the CHM gene of a Dutch patient diagnosed with choroideremia [CHM (MIM303100)], an X-linked progressive eye disorder. The L1 was integrated into exon 6 in reverse orientation, leading to aberrant splicing of the CHM mRNA (22). Interestingly, this L1 element, designated L1_CHM, appeared to be full-length, which prompted us to characterize it in detail. L1_CHM contains two 3'-transduced sequences that allowed us to infer its retrotransposition path. We also demonstrate that L1_CHM is highly active, but more importantly, we present data indicating that the actual L1 insertion into the CHM gene took place during early embryogenesis in the patient’s mother.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Retrotransposition path of L1_CHM
L1_CHM was PCR-amplified from genomic DNA of the CHM patient using primers flanking exon 6 of the CHM gene. Subsequent sequence analysis of the PCR product was performed with primers derived from a consensus sequence of active human L1 elements. L1_CHM has a length of 6688 bp and is surrounded by a perfect 14 bp TSD (5'-AGAAGATCAATTAG-3', designated TSD1 in Fig. 1). The 3'-UTR contains the ACA sequence (positions 5922–5924) diagnostic for the Ta subset (23). At its 3'-end, L1_CHM contains three poly(A) stretches of 60, 14 and 71 nucleotides, respectively, interrupted by transduced sequences of 119 and 406 bp (Fig. 1 and Supplementary Material, Fig. S1). These 3'-transductions are indicative of multiple rounds of retrotransposition.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Structure and origin of L1CHM. A schematic representation of L1CHM integrated in exon 6 of the CHM gene on chromosome Xq21 is shown on the top. The L1 element is surrounded by a 14 bp target site duplication (TSD1). The two ORFs (ORF1, ORF2) and three poly(A) stretches interrupted by two transduced sequences (positions 6079–6197 and positions 6212–6617) of L1CHM are indicated. In the middle, the 3'-UTR of L1CHM is expanded and aligned with an allelic variant of its precursor L1 in contig AC004200.1 on chromosome 6p21. This L1 element is flanked by a 15 bp target site duplication (TSD2). Possible pre-integration sites of the precursor to the L1CHM precursor on chromosome 10p15 and 18p11 are indicated at the bottom.

 
A transduced sequence acts as a molecular address that permits the identification of the precursor to the transduction event. A BLAST search (24) of the NCBI non-redundant nucleotide sequence database (http://www.ncbi.nih.gov) showed that the 406 bp transduction of L1_CHM is a precise match to sequences from BX005091 [GenBank] .7 (EMBL) and AC004200 [GenBank] .1 (GenBank) on chromosome 6p21. BX005091 [GenBank] .7 lacks an L1 element and represents the pre-integration site. A 6212 bp L1 is located immediately 5' of the 406 bp sequence in AC004200 [GenBank] .1 (Fig. 1). This L1 is identical in sequence to L1_CHM (1–6211) except for three additional guanine residues at its 5'-end, a single nucleotide change in ORF2 (T position 28 485 versus C in L1_CHM position 2439), and a poly(A) stretch of 58 instead of 60 adenosine residues. As in L1_CHM, a 119 bp transduced sequence followed by a poly(A) stretch of 14 nucleotides is present at the 3'-end. The L1 element is flanked by a perfect 15 bp TSD (5'-AAAAAACACAGTGAA-3', designated TSD2 in Fig. 1). Thus, the L1 in AC004200 [GenBank] .1 and the precursor to L1_CHM are likely to be alleles at a locus on chromosome 6p21, and this locus is polymorphic as to the presence or absence of an L1 element. Indeed, it has previously been established that an L1 element is present at this locus in 0 to 4% of alleles from different human populations (1,2).

A BLAT search (25) of the UCSC genome database [http://genome.ucsc.edu, Human May 2004 (hg17) assembly] using the 119 bp transduction of L1_CHM and its precursor produced exact matches near the telomeres of the short arms of chromosome 10 (10p15.3 nucleotides g.60 462–g.60 344) and chromosome 18 (18p11.32 nucleotides g.14 856–g.14 738). No full-length L1 element is present. Likewise, a BLAST search (24) of the NCBI non-redundant nucleotide sequence database (http://www.ncbi.nih.gov) revealed no sequences in which the 119 bp segment was preceded by an L1. Thus, the precursor to the L1_CHM precursor may reside on either chromosome 10pter or 18pter. PCR analysis of these regions in 200 Dutch individuals failed to detect an L1 element. This indicates that the presumed L1 element at 10p15 or 18p11 is very rare or no longer present in this population, although a technical problem cannot be excluded. Taken together, these findings indicate a scenario in which an L1 on chromosome 6p21 retrotransposed into the CHM gene on chromosome Xq21. The L1 element on chromosome 6p21 in turn is derived from either chromosome 10p15 or chromosome 18p11.

L1_CHM displays high retrotransposition activity
L1_CHM was tested in an established cultured cell assay that uses an enhanced green fluorescent protein (EGFP) marker to determine retrotransposition activity (1,26). L1_RP, an L1 element that was discovered as a de novo insertion into the retinitis pigmentosa 2 gene (27,28), served as a standard for comparison in this assay. The retrotransposition activity of L1_CHM was about one-third that of L1_RP, classifying L1_CHM as a highly active or ‘hot’ L1 (1). Hot L1s comprise only a small proportion of active L1s but are responsible for the majority of retrotransposition in the human population.

Somatic and germ-line mosaicism for L1_CHM
In order to determine when the L1 insertion into the CHM gene took place, the relatives of the CHM patient were studied. CHM exhibits an X-linked mode of inheritance. Males affected with CHM experience night blindness, followed by progressive visual field loss leading to tunnel vision and often blindness. These symptoms are paralleled by degeneration of the choroid and retina, beginning in the midperiphery and gradually progressing toward the macula (29). Figure 2 shows a fundus photograph of the patient (II.1) demonstrating the extensive chorioretinal hypopigmentation with preservation of pigment in the central macula that is typical for CHM. Female CHM carriers generally have no serious visual impairment. They can, however, be diagnosed by fundoscopic examination as they manifest patchy areas of chorioretinal degeneration resulting from X chromosome inactivation. Ophthalmological examination of the patient's mother (I.2) revealed three hyperpigmented radial lines in the temporal inferior part of the right fundus but was otherwise unremarkable (Fig. 2). One of the patient's sisters (II.2) showed normal optic discs, marked peripapillary atrophy in zones alpha and beta, a hypopigmented fundus with some white flecks in the periphery, slight pepper and salt pigment alterations in the peripheral retina and hyperpigmented equatorial rings and streaks (Fig. 2). She was diagnosed as a carrier. Apart from changes consistent with her myopia, the patient's other sister (II.3) had a normal fundus appearance (not shown).

View larger version (98K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Fundus images of the CHM patient (II.1), his mother (I.2) and carrier sister (II.2). Numbers correspond to the pedigree in Fig. 3A. The mother (I.2) has a normal fundus with a few choroidal hyperpigmentations in the midperiphery (arrowheads). The patient (II.1) shows the hallmarks of CHM: extensive midperipheral atrophy of the RPE and choroid, leading to a hypopigmented fundus while sparing its center till the end-stage, together with attenuated retinal vessels (arrowheads). Carrier female II.2 shows atrophy around the optic disc (left panel, arrowhead) and so-called pepper and salt pigment changes with hyperpigmentations in the equatorial region (right panel, arrowheads).

 
Initial genotyping of the family for two polymorphic markers within the CHM gene, i.e. a single-nucleotide polymorphism (c.351G/A) in exon 5 (30) and a microsatellite marker in intron 9 (31), showed that the patient and his two sisters share the disease haplotype (Fig. 3A). For female II.3, this was inconsistent with her clinical examination. Therefore, the presence of the L1_CHM insertion in the family members was investigated using a PCR assay that amplified the mutant CHM allele containing the L1_CHM insertion and the normal CHM allele. Both alleles were found in lymphocyte-derived DNA from the mother but the mutant PCR product had a significantly reduced level compared with the normal PCR product (Fig. 3B). These results indicate that the mother is a somatic mosaic for the L1 insertion. PCR analysis further showed that female II.2 is heterozygous for the L1 insertion, whereas female II.3 has only the normal CHM allele (Fig. 3B). The finding that female II.3 inherited the at-risk haplotype but not the L1_CHM insertion proves that the mother is a germ-line mosaic. Thus, these data demonstrate that the L1 retrotransposition took place in the patient's mother. A precursor to L1_CHM is actually present on one of her 6p21 alleles (Supplementary Material, Fig. S2). Since the patient's mother shows both somatic and germ-line mosaicism for the L1 insertion into the CHM gene, the L1 retrotransposition event must have occurred during early embryogenesis, before the germ-line segregated from the somatic lineages.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. Molecular analysis of the CHM family. (A) Pedigree and haplotype analysis for a single-nucleotide polymorphism (c.351G/A) in CHM exon 5 and a microsatellite marker in CHM intron 9. The at-risk haplotype is indicated in bold. (B) PCR analysis of genomic DNA from the family members using primers derived from CHM intron 6 and L1CHM to amplify the mutant allele and primers flanking CHM exon 6 to amplify the normal allele. Fragment sizes are indicated.

 
Garcia-Perez et al. (32) report that human embryonic stem cells express endogenous L1 elements and can accommodate L1 retrotransposition in vitro. In reinforcement of these data, our detailed analysis of a disease-causing L1 insertion provides in vivo evidence that L1 retrotransposition can occur very early in human embryonic development.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Molecular characterization of L1_CHM
L1_CHM was amplified from genomic DNA of the CHM patient with the Expand Long Template PCR system (Roche) using primers flanking exon 6 (5'-CGGAGGACTGGAATTTACTGCTTTATGA-3' and 5'-GAATTTCAGCTAATCAATTCTGAGCCTG-3') as described (22). The PCR product was purified with Qiaquick columns (Qiagen). Sequence analysis was performed using primers derived from a consensus sequence of active human L1 elements (adapted from 28). Primer sequences are available upon request. Sequencing reactions were carried out by the use of BigDye Terminator chemistry according to the guidelines provided by the manufacturer (PE Applied Biosystems) and analyzed on an ABI 3700 capillary DNA sequencer (PE Applied Biosystems).

Molecular analysis of possible L1_CHM precursor loci
To investigate the presence of a precursor L1 element at 10p15/18p11, a primer derived from the 3'-UTR of L1_CHM (5'-CCTAATGCTAGATGACACA-3') and a primer specific for both 10p15 and 18p11 (5'-AAATGCTTGAGAGGATGG-3') were used. The expected size of the PCR fragment is 446 bp. The empty allele at 10p15/18p11 was amplified using primers 5'-AAATGCTTGAGAGGATGG-3' and 5'-TGGGATGAGCAATGAATAAC-3' (272 bp product).

Retrotransposition assay
Primer 5'-ATTTCGCCGGCGTTGATTAGAAGATCAATTAGCCTCCTCGGTTC-3' containing a NotI site and primer 5'-ATACATATGTACACGGTACGACCACGCGA-3' containing a BstZ17I site were used for PCR amplification of L1_CHM from the patient's genomic DNA by Expand Long Template PCR (Roche) following the protocol provided by the manufacturer. The PCR product was digested with NotI and BstZ17I and swapped into pL1_RP–EGFP, a previously published construct containing L1_RP tagged with an EGFP retrotransposition cassette (1,18). Then pL1_CHM–EGFP was assayed for its ability to retrotranspose in cultured human 143B TK-osteosarcoma cells, as described (1,26), using pL1_RP–EGFP as a standard for comparison.

Clinical and molecular genetic analysis of the CHM family
The patient and his relatives were examined by fundoscopy and functional ophthalmological methods. Genomic DNA was isolated from blood lymphocytes by a salt extraction procedure (33). Genotyping for a single-nucleotide polymorphism (c.351G/A) in exon 5 (30) was carried out by PCR amplification with primers derived from intron 5 (5'-AGCCTGGTGTTTATTATTATATT-3') and intron 6 (5'-GCAAAGATGGGTAAAATTAGT-3'). PCR products were purified with Qiaquick columns (Qiagen) and sequenced as described above. Analysis of a microsatellite marker in intron 9 was performed using a 6-FAM-labeled forward primer (5'-TCAGTACAGTGCCTTGTAGTGGA-3') and an unlabeled reverse primer (5'-GTTGATATGTTATTAATACCAAGGAG-3') as described previously (31). To investigate the presence of the L1_CHM insertion, a PCR assay amplifying both the mutant allele containing L1_CHM and the normal CHM allele was designed. For the PCR amplification of mutant CHM allele primers derived from intron 6 (5'-CGGAGGACTGGAATTTACTGCTTTATGA-3') and the 5'-UTR of L1_CHM (5'-TGCTCTCTTCAAAGCTGTCAGACAG-3') were used, whereas the normal CHM allele was amplified with intronic primers flanking exon 6 (5'-CGGAGGACTGGAATTTACTGCTTTATGA-3' and 5'-GAATTTCAGCTAATCAATTCTGAGCCTG-3'). The PCR reaction mixture contained 1 x PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 400 nM of primers, 250 ng of genomic DNA and 1 unit Taq polymerase (Invitrogen). After 2 min at 94°C, 35 cycles were carried out consisting of 20 s at 94°C, 20 s at 56°C and 1 min at 72°C, followed by 2 min at 72°C. PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide.

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
We thank Dorien van de Pol, Krista Voesenek and Ellen Blokland for excellent technical assistance. We are also grateful to the patient and his family for participating in this study. Our work was supported by the kind donations of several Dutch organizations, i.e. Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Gelderse Blindenvereniging, Rotterdamse Vereniging Blindenbelangen, Stichting Blindenhulp, Stichting OOG and Stichting voor Ooglijders. This work was also supported by the European Union Research Training Network Grant RETNET MRTN-CT-2003-504003 (K.N., A.M.).

Conflict of Interest statement. None declared.

Note added in proof: Real-time Q-PCR analysis of genomic DNA from the mosaic mother I.2 and the choroideremia patient II.1 indicated that I.2 carries the L1_CHM insertion in 3% of her lymphocytes (Supplementary Material, Fig. S3).

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TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 

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