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Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene

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Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.
  SHORT COMMUNICATION Familial retinoblastoma due to intronic LINE-1insertion causes aberrant and noncanonical mRNAsplicing of the  RB1  gene Carlos Rodríguez-Martín 1 , Florencia Cidre 1 , Ana Fernández-Teijeiro 2 , Gema Gómez-Mariano 1 ,Leticia de la Vega 1 , Patricia Ramos 1 , Ángel Zaballos 3 , Sara Monzón 1,4 and Javier Alonso 1 Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in oneallele of the  RB1  gene have a high predisposition to develop RB. Here, we present the  󿬁 rst case of familial RB caused by a  de novo   insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the  RB1  gene that caused a highlyheterogeneous splicing pattern of  RB1  mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced bymassive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new  󿬁 ndingthat up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-basedstrategies have the potential to detect disease-causing transposon insertions. It also con 󿬁 rms that the incorporation of newgenetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique andexceptional genetic alterations. Journal of Human Genetics   advance online publication, 14 January 2016; doi:10.1038/jhg.2015.173 Retinoblastoma (RB, MIM 180200) is an embryonic neoplasm of retinal srcin with an incidence of 1 in 15 000 – 20 000 live births. 1,2 Approximately 40% of the patients harbor a constitutional mutationin one allele of the  RB1  gene, which predisposes to develop RB. 3 Here,we report an RB family with two affected members that harbor anexceptional mutational event in the  RB1  gene leading to hereditary RB.The proband and his father were diagnosed with bilateral RB at the ageof 4 and 18 months, respectively, and were referred to our laboratory to identify the mutation that predisposed to RB in this family.Research was approved by the institutional ethics committee of theInstituto de Salud Carlos III and written consent were obtained fromall members of the family. Proband ’ s blood DNA was  󿬁 rst screened formutations in  RB1  gene using a standard strategy (polymerase chainreaction (PCR) sequencing of all exons and promoter and multiplex ligation-dependent probe ampli 󿬁 cation), 4,5 but no causative mutationswere identi 󿬁 ed. Thus, we subsequently analyzed  RB1  mRNA isolatedfrom peripheral leukocytes to identify putative intronic mutations thatcould affect splicing. 6,7 RB1  cDNA was reverse transcription-PCR ampli 󿬁 ed in threeoverlapping fragments (exons 1 – 8, 7 – 17 and 16 – 27). As shown inFigure 1a, reverse transcription-PCR of exons 7 – 17 showed a highly heterogeneous aberrant splicing pattern produced by skipping of exons 14, 15 or 16 and/or inclusion of cryptic exons of variablelength (Figure 1b and Supplementary Figures S1A and H).BLAST analysis ( of the crypticexons showed a 98% homology of these sequences with the 5 ′  end of the long interspersed element-1 (LINE-1) transposons family  8 (Figure 1b).We next performed a PCR assay with primers localized in thecryptic exons (LINE-1 sequences) and in the exon 14 of the  RB1 (Figure 2a) to con 󿬁 rm at the genomic level this mutation. This assay rendered a speci 󿬁 c amplicon in the two affected members of thefamily (proband and his father) but not in the unaffected ones(proband ’ s brother and father ’ s parents), demonstrating that themutation was a  de novo  event occurring for the  󿬁 rst time in theproband ’ s father.To characterize the complete LINE-1 sequence inserted in the  RB1 gene, we performed a long-PCR assay from introns 13 to 15.Sequencing of the amplicon obtained by Roche/454 massive parallelsequencing con 󿬁 rmed that it contained a full-length LINE-1(6.044 bp) and adjacent intronic and exonic sequences derived from RB1  (Figure 2b). The complete sequence of the LINE-1 andsurrounding   RB1  exons has been deposited in GenBank databaseunder the accession number KU308246. According to this sequence 1 Unidad de Tumores Sólidos Infantiles, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain; 2 Unidad de Gestión Clínica Intercentros de Oncología Pediátricas, Hospitales Universitarios Virgen Macarena y Virgen del Rocío, National Reference Unit for Retinoblastoma,Sevilla, Spain;  3 Unidad de Genómica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain and  4 Centro de Investigación Biomédica en Red deEnfermedades Raras (CIBERER U758), Instituto de Salud Carlos III, Madrid, SpainCorrespondence: Dr J Alonso, Unidad de Tumores Sólidos Infantiles, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de SaludCarlos III (ISCIII), Ctra. Majadahonda-Pozuelo Km 2, Majadahonda, Madrid 28220, Spain.E-mail: fjalonso@isciii.esReceived 17 October 2015; revised 21 December 2015; accepted 25 December 2015  Journal of Human Genetics (2016),  1 – 4 &  2016 The Japan Society of Human Genetics All rights reserved 1434-5161/16  the LINE-1 was inserted at position chr13:48954187_48954188(GRCh37/hg19 assembly), disrupting the intron 14/exon 15 boundary of the  RB1  gene (NM_000321.2:c.1390-2insL1) (Supplementary Figure S2).We performed an  in silico  analysis using L1Xplorer tool( 9 tocharacterize the functional elements of LINE-1 in detail. This analysisshowed that 24 out of 25 parameters analyzed were conserved(Figure 2c), including the most characteristic domains of LINE-1 thatare key for its function (5 ′ -untranslated region, open reading frame-1and open reading frame-2, the 66-bp intergenic spacer, a polyadenyla-tion signal and a 33 bp 3 ′ -A-tail). 10 This analysis suggest that thisLINE-1 could potentially remain active and be capable of secondary retrotransposition events, as it has been previously reported in twoother cases of full-length LINE-1 insertions ( β -globin gene andretinitis pigmentosa-2 gene). 11 In agreement with this, the LINE-1inserted in the  RB1  gene was highly homologous to one of the mostactive LINE-1s (hot LINE-1s) described in humans 12 (Figure 2c). An Figure 1  Characterization of retinoblastoma 1 ( RB1 ) mRNA aberrant splicing in the retinoblastoma patient. ( a ) RNA isolated from peripheral blood leukocytesof the proband and his affected father were ampli 󿬁 ed by reverse transcription-polymerase chain reaction (RT-PCR) in three overlapping fragments. Ampliconsobtained were then analyzed by gel electrophoresis, cloned in pGEM-T vector and sequenced to identify aberrant mRNA products. No aberrant mRNAs wereobserved in fragments covering exons 1 – 8 and exons 16 – 27. By contrast, 20% (11 out of 54) of the clones derived from cDNA fragments covering exons7 – 17 showed an aberrant pattern. The  󿬁 gure shows representative PCR-ampli 󿬁 ed inserts derived from clones covering exons 7 – 17. Inserts which sizesdiffered from the expected correspond to aberrant mRNAs and are marked with an arrow. ( b ) Scheme of the different types of aberrant  RB1  mRNAsidenti 󿬁 ed. The aberrant transcripts were produced by skipping of exons 14, 15 or 16 (gray boxes) or by integration of new cryptic exons (CE, black boxes) ofvariable length (89, 95, 103 and 106 bp), with or without additionally skipped exons. All cryptic exons shared a 89-nucleotide common sequence but haddifferent 5 ′  end sequences. Each aberrant mRNA was predicted to codify a non-functional RB1 protein (i.e. truncated proteins with premature stop codons oraberrant mRNAs lacking in-frame exon 14). BLAST analysis of the cryptic exon sequences showed a high homology with the long interspersed element-1(LINE-1) consensus sequence. LINE-1 retrotransposition causes familial retinoblastoma C Rodríguez-Martín  et al  2  Journal of Human Genetics  Figure 2  Comprehensive genomic characterization of long interspersed element-1 (LINE-1) retrotransposition in the retinoblastoma 1 ( RB1 ) gene and itseffects on consensus splicing motifs. ( a ) A PCR assay using a forward primer localized in intron 13 of  RB1 , close to exon 14, and a reverse primer derivedfrom the LINE-1 fragment integrated in the aberrant transcripts, was designed to con 󿬁 rm the insertion of the LINE-1 at the genomic level. An amplicon withthe expected size (arrowhead on the left) was detected in the affected members of the family (the proband and his father), but was not observed in theunaffected members (a proband ’ s brother and the father ’ s parents), indicating that this assay has diagnostic value. An unspeci 󿬁 c amplicon (asterisk on theright) was observed in all DNA analyzed. DNA from an unrelated individual was used as control. ( b ) The scheme shows the complete genomic structure ofthe LINE-1 insertion in the context of the  RB1  gene. To obtain the complete sequence of the LINE-1, a long-PCR assay from intron 13 – 15 of  RB1  gene wasperformed. Next, the 6.7 kb amplicon obtained was sequenced by Roche/454 massive parallel sequencing. A total of 24 723 reads were obtained andassembled using gsAssembler v.2.8 software (Roche, Branford, CT, USA), obtaining a unique contig of 6655 bp, with a 243x median depth per nucleotide.This contig contained a full-length LINE-1 (6.044 bp) that conserved the key LINE-1s functional domains. Tandem segmental duplication (TSD) 8 repeatscharacteristic of LINE-1 retrotransposition are also shown. ( c ) DNA phylogenetic analysis (ClustalW-Phylogeny, 18 carried out with thesix more active LINE-1s in the human genome (hot LINE-1s) 12 and the LINE-1 characterized in this work. The new LINE-1 inserted in the  RB1  gene washighly homologous to a LINE-1 located in chromosome 6p21 (NCBI accession number AC004200.1). Percent identity between both LINE-1s is shown.Transposon activity as reported in Brouha  et al. 12 is speci 󿬁 ed. Intactness score indicates the number of LINE-1 functional parameters tested by theL1Xplorer tool that are conserved versus the total of parameters analyzed. ( d ) Scheme representing the splicing motifs and their location before and afterLINE-1 retrotransposition. Branch points (BP), Polypyrimidine tracks ((Py)n) and acceptor/donor splice sites (ASS, DSS) are indicated. Exonized sequencesare shown in capital letters. BP and splice sites scores were calculated with Human Splice Finder tool and showed within parentheses when available. LINE-1 retrotransposition causes familial retinoblastoma C Rodríguez-Martín  et al  3  Journal of Human Genetics  alignment of both sequences with indication of the changes observedis shown in Supplementary Figure S3.In an attempt to explain the different aberrant mRNAs observed, weanalyzed in detail the impact of LINE-1 insertion on the consensussequences involved in mRNA processing using the Human SpliceFinder tool (HSF) ( 13 Since the LINE-1 wasinserted between the polypyrimidine track located in intron 14 and theAG dinucleotide of the acceptor site of exon 15, LINE-1 sequencesreplaced the original branch point and polypyrimidine track (Figure 2d).  In silico  analysis of the new context showed a new branchpoint with a high score (81.56) provided by the LINE-1 sequence thatcould be used during splicing of exon 15. However, a consensuspolypyrimidine track was not found, which is expected to severely affect exon recognition. In agreement with this, nearly 50% of theaberrant mRNA species identi 󿬁 ed lacked exon 15.As a consequence of the LINE-1 transposition, the polypyrimidinetrack and the high score branch point site situated in intron 14 werethen placed upstream the LINE-1 itself (Figure 2d). Consequently, thecryptic exons detected used three noncanonical acceptor splice sites(AT, AT and CG) derived from intron 14 of   RB1  and one canonicalacceptor splice site (AG) situated in the LINE-1 sequence (HSF score75.42). Of note, although the canonical splice site was disrupted, thesrcinal branch point and polypyrimidine track placed upstreamLINE-1 transposon were still strong enough to provoke the exoniza-tion of intronic/LINE-1 sequences. All cryptic exons used a singlecanonical GT donor splice site located at position 97 of the LINE-1consensus sequence (HSF score 75.49). This splice donor site was alsoobserved in two cases of partial LINE-1 exonization in genes  ABHD5 and  NF1  (neuro 󿬁 bromatosis type I). 14,15 The RB case reported here indicates that while standard mutationalscreening are effective to detect the majority of the mutations causing familial RB, 16 ad hoc   strategies should be achieve to resolve some cases.This study exempli 󿬁 ed how an RNA-based mutation analysis incombination with massive parallel sequencing may be an effectivemethod to identify retrotransposon insertions. In this line, all 18pathogenic insertions of LINE-1s and Alu elements in the  NF1  genereported by Wimmer  et al. 15 were identi 󿬁 ed because they alteredtranscripts splicing.We reported here the  󿬁 rst case of familial RB caused by retrotransposition of a LINE-1 into  RB1  gene and provided acomprehensive analysis of the effects caused by this LINE-1 insertionon  RB1  mRNA splicing. Interestingly, until now only three cases of LINE-1 insertions have been associated to familial cancer: a case of familial adenomatous polyposis 17 and two cases of NF1, 15,17 whichsuggest that these mutations are probably underrepresented becausethese events may be overlooked by the most commonly used mutationdetecting methods that rely on PCR ampli 󿬁 cation of small amplicons(i.e. exons from genomic DNA). We propose that in some cases acombination of RNA-based strategies and massive parallel sequencing can be useful to identify and characterize the causative mutation inhereditary diseases. The characterization of these rare events can helpto design speci 󿬁 c PCR assays to screen the presence of the mutation inthe family, providing a simple genetic test for future screening of other 󿬁 rst degree relatives (including prenatal testing). Nucleotide data  The nucleotide sequence data reported is available in the GenBank database under the accession number KU308246. CONFLICT OF INTEREST The authors declare no con 󿬂 ict of interest. ACKNOWLEDGEMENTS This study was funded by grants of the Instituto de Salud Carlos III(PI12/00816 and RTICC RD12/0036/0027). CR-M was supported by aMINNECO contract. FC was supported by Asociación Pablo Ugarte andMiguelañez SA. SM was supported by a CIBERER contract. We greatly appreciate the collaboration of the RB patients, their parents and their families. 1 Lohmann, D. Retinoblastoma.  Adv. Exp. Med. Biol.  685 , 220 – 227 (2010).2 Dimaras, H., Kimani, K., Dimba, E. A., Gronsdahl, P., White, A., Chan, H. S.  et al. Retinoblastoma.  Lancet   379 , 1436 – 1446 (2012).3 Valverde, J. R., Alonso, J., Palacios, I. & Pestana, A. RB1 gene mutation up-date, ameta-analysis based on 932 reported mutations available in a searchable database. BMC Genet.  6 , 53 (2005).4 Alonso, J., Garcia-Miguel, P., Abelairas, J., Mendiola, M., Sarret, E., Vendrell, M. T. et al.  Spectrum of germline RB1 gene mutations in Spanish retinoblastoma patients:phenotypic and molecular epidemiological implications.  Hum. Mutat.  17 ,412 – 422 (2001).5 Parsam, V. L., Kannabiran, C., Honavar, S., Vemuganti, G. K. & Ali, M. J. Acomprehensive, sensitive and economical approach for the detection of mutations inthe RB1 gene in retinoblastoma.  J. Genet.  88 , 517 – 527 (2009).6 Parsam, V. L., Ali, M. J., Honavar, S. G., Vemuganti, G. K. & Kannabiran, C. Splicingaberrations caused by constitutional RB1 gene mutations in retinoblastoma.  J. Biosci. 36 , 281 – 287 (2011).7 Dehainault, C., Michaux, D., Pages-Berhouet, S., Caux-Moncoutier, V., Doz, F.,Desjardins, L.  et al.  A deep intronic mutation in the RB1 gene leads to intronicsequence exonisation.  Eur. J. Hum. Genet.  15 , 473 – 477 (2007).8 Ostertag, E. M. & Kazazian, H. H. Jr. Biology of mammalian L1 retrotransposons.  Annu.Rev. Genet.  35 , 501 – 538 (2001).9 Penzkofer, T., Dandekar, T. & Zemojtel, T. L1Base: from functional annotation toprediction of active LINE-1 elements.  Nucleic Acids Res.  33 , D498 – D500 (2005).10 Beck, C. R., Garcia-Perez, J. L., Badge, R. M. & Moran, J. V. LINE-1 elements instructural variation and disease.  Annu. Rev. Genomics Hum. Genet.  12 ,187 – 215 (2011).11 Kimberland, M. L., Divoky, V., Prchal, J., Schwahn, U., Berger, W. & Kazazian, H. H. Jr.Full-length human L1 insertions retain the capacity for high frequency retrotransposi-tion in cultured cells.  Hum. Mol. Genet.  8 , 1557 – 1560 (1999).12 Brouha, B., Schustak, J., Badge, R. M., Lutz-Prigge, S., Farley, A. H., Moran, J. V.  et al. Hot L1s account for the bulk of retrotransposition in the human population.  Proc. Natl Acad. Sci. USA  100 , 5280 – 5285 (2003).13 Desmet, F. O., Hamroun, D., Lalande, M., Collod-Beroud, G., Claustres, M. & Beroud,C. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res.  37 , e67 (2009).14 Samuelov, L., Fuchs-Telem, D., Sarig, O. & Sprecher, E. An exceptional mutationalevent leading to Chanarin – Dorfman syndrome in a large consanguineous family.  Br. J.Dermatol.  164 , 1390 – 1392 (2011).15 Wimmer, K., Callens, T., Wernstedt, A. & Messiaen, L. The NF1 gene contains hotspotsfor L1 endonuclease-dependent de novo insertion.  PLoS Genet.  7 , e1002371 (2011).16 Price, E. A., Price, K., Kolkiewicz, K., Hack, S., Reddy, M. A., Hungerford, J. L.  et al. Spectrum of RB1 mutations identi 󿬁 ed in 403 retinoblastoma patients.  J. Med. Genet. 51 , 208 – 214 (2014).17 Miki, Y., Nishisho, I., Horii, A., Miyoshi, Y., Utsunomiya, J., Kinzler, K. W.  et al. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in acolon cancer.  Cancer Res.  52 , 643 – 645 (1992).18 Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A.,McWilliam, H.  et al.  Clustal W and Clustal X version 2.0.  Bioinformatics   23 ,2947 – 2948 (2007). Supplementary Information accompanies the paper on Journal of Human Genetics website ( LINE-1 retrotransposition causes familial retinoblastoma C Rodríguez-Martín  et al  4  Journal of Human Genetics
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