Sva retrotransposon insertion in exon of mmr genes results in aberrant rna splicing and causes lynch syndrome

ABSTRACT Lynch syndrome is an autosomal dominant hereditary cancer syndrome in which many cancers develop, the main one being colorectal cancer. Germline pathogenic variants in one of four

mismatch repair (MMR) genes are known to be causative of this disease. Accurate diagnosis using genetic testing can greatly benefit the health of those affected. Recently, owing to the

improvement of sequence techniques, complicated variants affecting the functions of MMR genes were discovered. In this study, we analyzed insertions of a retrotransposon-like sequence in

exon 5 of the _MSH6_ gene and exon 3 of the _MSH2_ gene found in Japanese families suspected of having Lynch syndrome. Both of these insertions induced aberrant splicing, and these variants

were successfully identified by mRNA sequencing or visual observation of mapping results, although a standard DNA-seq analysis pipeline failed to detect them. The insertion sequences were

~2.5 kbp in length and were found to have the structure of an SVA retrotransposon (SVA). One SVA sequence was not present in the hg19 or hg38 reference genome, but was in a Japanese-specific

reference sequence (JRGv2). Our study illustrates the difficulties of identifying SVA insertions in disease genes, and that the possibility of polymorphic insertions should be considered

when analyzing mobile elements. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS _TP53_ MINIGENE ANALYSIS OF 161 SEQUENCE

CHANGES PROVIDES EVIDENCE FOR ROLE OF SPATIAL CONSTRAINT AND REGULATORY ELEMENTS ON VARIANT-INDUCED SPLICING IMPACT Article Open access 08 May 2025 A 39 KB STRUCTURAL VARIANT CAUSING LYNCH

SYNDROME DETECTED BY OPTICAL GENOME MAPPING AND NANOPORE SEQUENCING Article Open access 29 November 2023 WIDESPREAD SOMATIC L1 RETROTRANSPOSITION IN NORMAL COLORECTAL EPITHELIUM Article Open

access 10 May 2023 INTRODUCTION Lynch syndrome is an autosomal dominant hereditary cancer syndrome in which colon cancer is particularly prominent. It is caused by a pathogenic variant in

one of the mismatch repair genes (MMR genes; _MLH1_, _MSH2_, _MSH6_, or _PMS2_) [1,2,3,4,5]. In recent years, owing to the advent of next-generation sequencing (NGS) and the progression of

analytical techniques, many variants affecting the functions of MMR genes have been revealed [6]. Lynch syndrome accounts for about 3% of all colon cancer cases [7, 8] and its prevalence in

the general population has been estimated at 1:440 [9]. Because various cancers besides colon cancer occur in those affected by this syndrome, determination of the responsible variant and

appropriate surveillance are important to the healthcare of patients [10]. Retrotransposons are “mobile genetic elements” that move in the genome. Transposon insertion causes a change in the

gene at or near the insertion point. Such a change is considered to provide room for the evolution of the genome [11]. Retrotransposons occupy ~40% of the human genome sequence and are

classified into two groups: long terminal repeat (LTR) and non-LTR. LTR retrotransposons do not have transposable element activity, while non-LTR retrotransposons, including LINE-1

(constitute 16.9% of the human genome), Alu (10.6%), and SVA (0.2%) elements, have such activity; this latter group covers approximately one-third of the human genome and causes various

hereditary diseases via its insertion [12]. SVA retrotransposons, hominid-specific retrotransposons, consist of SINE (short interspersed repetitive elements), VNTR (variable number of tandem

repeats), and Alu, and are ~3 kb in length. This type of retrotransposon is rare, with ~2700 copies of it in the human genome [13]. Regarding its relationship with disease, it was reported

that a cause of Fukuyama-type congenital muscular dystrophy was splice abnormality by the insertion of an SVA-type retrotransposon [14]. With regard to Lynch syndrome, an Alu insertion

variant in MSH2 was reported [15, 16], while a report of a single case of SVA insertion in _PMS2_ was also published [17]. In this study, insertion of an SVA-type retrotransposon was found

in exonic regions of the _MSH2_ or _MSH6_ gene. One of the inserted sequences aligned only with JRGv2 (Japanese Reference Genome V2) provided by the Tohoku Medical Megabank but not with

standard human reference genomes. RNA sequencing revealed an aberrant alternative splicing event associated with these variants. MATERIALS AND METHODS PATIENTS Patients enrolled in Study for

the Establishment of Effective Screening and Diagnosis of Lynch Syndrome (Dial study) were analyzed. Case 1 (Fig. 1a) had never been diagnosed with cancer. However, his father had developed

three metachronous Lynch syndrome-associated cancers (colorectal, ureteral, and bladder cancers). Microsatellite instability (MSI) and immunohistochemical testing revealed that the ureteral

and bladder cancers showed microsatellite instability-high (MSI-H) and loss of MSH6. Case 2 (Fig. 1b) was diagnosed with metachronous multiple cancers and his older brother showed a similar

phenotype. In both siblings, the colorectal cancer was MSI-H, and loss of MSH2/MSH6 was observed (Fig. 1b, III.2, 3). All procedures were performed in accordance with the ethical standards

of the responsible committee on human experimentation and with the 1964 Helsinki Declaration, as revised in 2013, as well as the Japanese ethical guidelines for human genome/gene analysis

research. This study was approved by the Institutional Review Boards of Saitama Cancer Center (no. 729). Written consent was obtained from the patient before inclusion in the study. DNA AND

RNA EXTRACTION FROM PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected in heparinized vacutainer tubes using

Ficoll®-Paque Premium (GE Healthcare, Chicago, IL, USA). These cells were resuspended in KBM502 (KOHJIN BIO, Sakado, Japan) supplemented with 10% FBS (GE Healthcare) and

penicillin–streptomycin (FUJIFILM Wako Pure Chemical, Osaka, Japan), and plated and cultured in tissue culture tubes (TPP, Trasadingen, Switzerland) at 37 °C in a 5% CO2 humidified

atmosphere. After 1 week, cells were divided into culture tubes for DNA extraction and RNA extraction with or without puromycin (Thermo Fisher Scientific, Waltham, MA, USA) treatment, as

reported previously [18]. DNA and RNA (with/without puromycin treatment) were extracted using AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer’s

instructions. NEXT-GENERATION SEQUENCING ANALYSIS FOR GENETIC TESTING Genetic testing for LS was conducted with both DNA and RNA. DNA was sequenced using QIAseq Targeted DNA Custom Panel

(Qiagen) including MMR genes (_MLH1_, _MSH2_, _MSH6_, _PMS2_, and _EPCAM_), in accordance with the manufacturer’s instructions. Transcripts of MMR genes were amplified by PCR with cDNA

synthesized from RNA and sequenced using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA), in accordance with the manufacturer’s instructions with slight modifications. [The

library amplification was carried out using KAPAHiFi DNA Polymerase (Kapa Biosystems, Wilmington, DE, USA), not NPM.] Sequencing was performed on Miseq (Illumina). The sequence reads were

analyzed with CLC Genomics Workbench (Qiagen, RRID: SCR_011853) using hg19 as a reference. The Japanese reference genome was obtained from JMORP (https://jmorp.megabank.tohoku.ac.jp/, [19]).

The accession numbers for the _MSH2_ and _MSH6_ genes (transcripts) were NG_007110.2 (LRG218t1) and NG_007111.1 (LRG219t1), respectively. Exons are numbered according to the accession

number of LRG. To confirm the inserted sequences, amplified PCR products were sequenced using the same method as in the RNA sequencing. Because there was difficulty amplifying the insertion

sequence of Case 2 using standard PCR enzyme, we used PrimeSTAR GXL DNA Polymerase (Takara Bio, Kusatsu, Japan). The list of primers designed for the amplification of MMR transcripts and

confirmation of the inserted sequences is given in Table 1. Visualization of mapping results was performed using Integrative Genomics Viewer software

(http://software.broadinstitute.org/software/igv/, RRID: SCR_011793). Sequence data resulted from this study is already submitted to the DNA DataBank of Japan (DDBJ) repository, accession

DRA009831 and DRA009891. RESULTS FINDING OF INSERTION IN EXONIC REGIONS OF _MSH6_ Multigene panel testing on the DNA sample of Case 1 using our standard sequencing pipeline detected neither

a pathogenic single-nucleotide variant nor copy number variation. However, sequencing of the _MSH6_ transcript revealed a deletion of the 5′ region of exon 5 in 27% of the reads of

transcripts isolated from puromycin treated PBMCs (23% in untreated cells) (Fig. 2a), suggesting that aberrant splicing was induced by the use of a cryptic 3′ splice site. Via careful

evaluation of the mapping of the DNA sequence around this region, we found a soft-clipped sequence in the middle of exon 5 consisting of repetitive “GGGAGA” units and accounted for 304 out

of a total of 749 reads covering this site (Fig. S2a). These results suggested the presence of a larger inserted sequence with the sequence at the 3′ end although the 5′ end of the insertion

had not been detected. According to this assumption, we attempted to amplify the inserted DNA fragment using a forward primer (_MSH6_ F) located in exon 4 together with an

insertion-specific reversed primer (_MSH6_ R) (Table 1, Fig. 3a). Since the wild-type sequence between this primer pair is 2.6 kb, amplification of an ~5 kb PCR fragment revealed that the

inserted sequence is ~2.4 kb (Fig. 3c). INSERTION OF SVA-TYPE RETROTRANSPOSON CAUSES ABERRANT SPLICING To examine the inserted fragment, we sequenced the whole 5 kbp amplicon as described in

“Materials and methods” section. The results revealed the presence of an insertion sequence in exon 5 with a target site duplication starting at a poly-T tract (Fig. S1a). Together with the

“GGAGA” repeats at the 3′ end, this suggested that the insertion resulted from retrotransposition of an SVA element into exon 5 (Fig. S1b). The characteristics of this sequence are

reminiscent of an SVA-type retrotransposon (SVA). However, our standard mapping method failed to pinpoint the position in the reference genome, probably because of the repetitive sequence.

Using de novo assembly software, we obtained the whole sequence of the insert. By re-mapping on the reference genome, the sequence starting from poly-T turned out to be unique to chr12:

96233959–96236309 and this region was annotated as SVA E by Repeat Masker (http://www.repeatmasker.org/). This variant was considered to be represented as NC_000002.11:

g.48030698_48030699ins[SVA;48030684_48030698]. In addition, detailed RNA analysis revealed the deletion of the initial 174 bp sequence of exon 5, which caused an in-frame deletion of the

MSH6 protein. Taking these findings together, we concluded that the insertion of a ~2.4 kbp SVA E retrotransposon into exon 5 changes its splicing acceptor site (Fig. 4a, Table 2). SVA

INSERTION MAY BE A MORE FREQUENT CAUSE OF LYNCH SYNDROME THAN WE ASSUMED As described above, aberrant splicing induced by SVA insertion has been reported for _PMS2_. Case 1 in this study led

us to the assumption that SVA insertion is not a very rare causes of Lynch syndrome. Thus, we performed a thorough investigation of cases in which one of the Lynch syndrome genes was

apparently mutated, but the nature of the variant had not been determined. The RNA sequence of _MSH2_ from Case 2 showed an aberrant transcript lacking 88 bp in the middle of exon 3 (Fig. 

2b). Carefully evaluating the read mapping exon 3 revealed soft-clipped sequence similar to Case 1 consisting of repetitive “GGGAGA” (234 out of a total of 406 reads) and poly-T tract (234

out of 394 reads) (Fig. S2b). We assumed that this insertion sequence was also an SVA retrotransposon, but this insertion was not amplified by our standard PCR reactions. Therefore, we

employed PrimeSTAR GXL DNA polymerase (Takara Bio) instead of our standard PCR polymerase EX Taq (Takara Bio) to achieve better extension of “difficult to replicate” regions such as AT- or

GC-rich regions and attempted both two-step and three-step PCR cycles with two kinds of primer pairs, _MSH2_ F1/R1 and _MSH2_ F2/R2 (Table 1, Fig. 3b). Then, the insert was successfully

amplified (Fig. 3d) and the product was subjected to NGS. Unfortunately, our de novo assembly program failed to construct a single sequence as in Case 1. Therefore, we manually created

contigs from adjacent reads and merged them into a single sequence. However, we could not map the merged sequence on either hg19 or hg38. Only a slightly similar sequence in chromosome 16 of

hg19 was present (Fig. S3a). We thus assumed that the sequence is specific to the Japanese. By searching GGGenome (https://gggenome.dbcls.jp/en/), the sequence was revealed to be unique to

chr6: 111170450–111172854 of JRGv2, a Japanese-specific reference sequence [19] (Fig. S3b). Coverage analysis using the JRGv2 decoy sequence also suggested that this insertion sequence may

be specific to the sequence (Fig. S4). Furthermore, this sequence was thought to be inserted into NC_000006.11:g.111278198_111278199 in hg19 (Fig. S5). This sequence was annotated as SVA F

by Repeat Masker. Thus, in Case 2, an SVA F retrotransposon was integrated into exon 3 of _MSH2_ and created an extra intron in the middle of this exon (Fig. 4b). This variant was considered

to be represented as NC_000002.11: g.47637427_47637428ins[SVA;47637413_47637427]. As a result, shorter mRNA causing a frameshift was formed (Table 2). DISCUSSION In the genetic testing of

patients suspected of having Lynch syndrome in a multi-facility collaborative investigation, we found two novel exonic insertions of SVA in _MSH2_ and _MSH6_, and showed the splice effect of

these variants. Insertion of SVA has been reported only in an intron of the PMS2 gene [17], so this study is the first to identify it in other MMR genes and in an exonic region of these

four genes. As to the final evaluation of the pathogenicity of the two variants against Lynch syndrome, that in Case 1 is a variant of uncertain significance (VUS; PM2: absent in population

data, PM4: protein-length-changing variant) and that in Case 2 is likely pathogenic (PVS1: predicted null variant, PM2: absent in population data), according to ACMG criteria [20]. Regarding

Case 1, we tried to determine that the variant was pathogenic or likely pathogenic because it is a long deletion variant of 58 amino acids, but the variants registered in InSiGHT

(http://insight-database.org/) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) did not have an in-frame deletion variant that was determined to be pathogenic or likely pathogenic within

these 58 amino acids. For the diagnosis of Lynch syndrome, molecular tests, such as MSI testing and immunohistochemistry (IHC), are widely used, but those results are not included in the

criteria. In recent years, evaluation methods that include the results of MMR gene variants of a tumor and the results of IHC have been advocated [21, 22]. Our results indicate the

difficulty in finding SVA insertions (and possibly any other insertions of DNA sequence larger than a few kilobases) by standard methods of genetic testing. We had problems detecting SVA

insertion in both Case 1 and Case 2. In Case 1, our standard DNA analysis failed to detect the abnormality. Only a special tool, such as Scramble (https://github.com/GeneDx/scramble), could

detect this change (Table S1). However, we were able to notice the abnormality by analyzing RNA, which prompted us to re-examine the result of DNA sequencing. The DNA change was barely

detectable by visual inspection of the mapping results. This supports the idea that RNA analysis is often useful to discover variants that are difficult to detect and evaluate using DNA [23,

24]. However, RNA analysis does not always work well. In Case 2, quite a few reads (6%) supportive of aberrant splicing were detected in the RNA derived from PBMCs with puromycin treatment

(no reads in untreated cells). The ratio of aberrant-splicing reads was also similar in a relative (Fig. 1b, III-2), but was not recognized as an aberrant change by our standard

computational analysis. Because the loss of MSH2 protein was observed in IHC, we examined the whole region of this gene by manually viewing the mapping and eventually discovered SVA

insertion. It is unclear why the ratio of variant transcripts was low, but here are a various possible causes, including technical problems [25, 26]. In view of the difficulty of detection,

it is possible that there are a significant number of hidden Lynch syndrome patients who harbor an insertion of SVA (or any fragment extending a few kilobases) in any of the genes causative

of this condition. In support of this idea, variants involving the insertion of a mobile element such as Alu and LINE-1 sequences have been reported in genes causative of the hereditary

cancer-predisposing syndrome including NF1 and BRCA [27, 28]. Another problem that we encountered is that the insertion sequence of Case 2 did not exist in hg19 or hg38, and existed only in

JRGv2, the Japanese reference genome. Active retrotransposons are known to generate polymorphic insertions by themselves [29, 30]. In addition, it has been reported that an Alu sequence

moves to another locus once per 20 births [31]. Our data and these studies suggested that the consideration of ethnic and individual differences is important for identifying the origin of

inserted mobile elements. In this study, we detected insertions of SVA in exons of the _MSH2_ and _MSH6_ genes. To date, we have identified 137 pathogenic or likely pathogenic variants and

65 variants of uncertain significance (VUS) from 580 probands suspected of Lynch syndrome based on family history and molecular testing. In light of the difficulty in detecting them, the

insertion of mobile elements including SVA may not be a rare cause of Lynch syndrome. In addition, our results indicate that RNA analysis helps to increase the possibility of detection,

although it is not sufficient to detect all kinds of structural variants. To achieve precise diagnoses of genetic disorders and provide appropriate surveillance/treatment to patients,

technological advances to detect currently undetectable (or hardly detectable) variants are awaited. REFERENCES * Leach FS, Nicolaides NC, Papadopoulos N, Liu B, Jen J, Parsons R, et al.

Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell. 1993;75:1215–25. Article  CAS  Google Scholar  * Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe

MK, et al. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature. 1994;368:258–61. Article  CAS  Google Scholar  *

Nicolaides NC, Papadopoulos N, Liu B, Wei YF, Carter KC, Ruben SM, et al. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature. 1994;371:75–80. Article  CAS 

Google Scholar  * Papadopoulos N, Nicolaides NC, Wei YF, Ruben SM, Carter KC, Rosen CA, et al. Mutation of a mutL homolog in hereditary colon cancer. Science. 1994;263:1625–9. Article  CAS 

Google Scholar  * Miyaki M, Konishi M, Tanaka K, Kikuchi-Yanoshita R, Muraoka M, Yasuno M, et al. Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nat

Genet. 1997;17:271–2. Article  CAS  Google Scholar  * Espenschied CR, LaDuca H, Li S, McFarland R, Gau CL, Hampel H, et al. Multigene panel testing provides a new perspective on Lynch

syndrome. J Clin Oncol. 2017;35:2568–75. Article  CAS  Google Scholar  * Lynch HT, de la Chapelle A. Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet. 1999;36:801–18.

CAS  PubMed  PubMed Central  Google Scholar  * Cunningham JM, Kim CY, Christensen ER, Tester DJ, Parc Y, Burgart LJ, et al. The frequency of hereditary defective mismatch repair in a

prospective series of unselected colorectal carcinomas. Am J Hum Genet. 2001;69:780–90. Article  CAS  Google Scholar  * Chen S, Wang W, Lee S, Nafa K, Lee J, Romans K, et al. Prediction of

germline mutations and cancer risk in the Lynch syndrome. JAMA. 2006;296:1479–87. Article  CAS  Google Scholar  * Dominguez-Valentin M, Sampson JR, Seppälä TT, Ten Broeke SW, Plazzer JP,

Nakken S, et al. Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database. Genet Med.

2020;22:15–25. Article  CAS  Google Scholar  * Kazazian HH Jr, Moran JV. Mobile DNA in Health and Disease. N. Engl J Med. 2017;377:361–70. Article  CAS  Google Scholar  * Cordaux R, Batzer

MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10:691–703. Article  CAS  Google Scholar  * Hancks DC, Kazazian HH Jr. SVA retrotransposons: evolution and

genetic instability. Semin Cancer Biol. 2010;20:234–45. Article  CAS  Google Scholar  * Taniguchi-Ikeda M, Kobayashi K, Kanagawa M, Yu CC, Mori K, Oda T, et al. Pathogenic exon-trapping by

SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature. 2011;478:127–31. Article  CAS  Google Scholar  * Kloor M, Sutter C, Wentzensen N, Cremer FW, Buckowitz A, Keller M, et

al. A large MSH2 Alu insertion mutation causes HNPCC in a German kindred. Hum Genet. 2004;115:432–8. Article  CAS  Google Scholar  * Baert-Desurmont S, Coutant S, Charbonnier F, Macquere P,

Lecoquierre F, Schwartz M, et al. Optimization of the diagnosis of inherited colorectal cancer using NGS and capture of exonic and intronic sequences of panel genes. Eur J Hum Genet.

2018;26:1597–602. Article  CAS  Google Scholar  * van der Klift HM, Tops CM, Hes FJ, Devilee P, Wijnen JT. Insertion of an SVA element, a nonautonomous retrotransposon, in PMS2 intron 7 as a

novel cause of Lynch syndrome. Hum Mutat. 2012;33:1051–5. Article  Google Scholar  * Nomura S, Sugano K, Kashiwabara H, Taniguchi T, Fukayama N, Fujita S, et al. Enhanced detection of

deleterious and other germline mutations of hMSH2 and hMLH1 in Japanese hereditary nonpolyposis colorectal cancer kindreds. Biochem Biophys Res Commun. 2000;271:120–9. Article  CAS  Google

Scholar  * Nagasaki M, Kuroki Y, Shibata TF, Katsuoka F, Mimori T, Kawai Y, et al. Construction of JRG (Japanese reference genome) with single-molecule real-time sequencing. Hum Genome Var.

2019;6:27. https://doi.org/10.1038/s41439-019-0057-7. Article  PubMed  PubMed Central  Google Scholar  * Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and

guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.

Genet Med. 2015;17:405–24. Article  Google Scholar  * Gray PN, Tsai P, Chen D, Wu S, Hoo J, Mu W, et al. TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the

detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome. Oncotarget. 2018;9:20304–22. Article  Google Scholar  * Shirts BH, Konnick

EQ, Upham S, Walsh T, Ranola JMO, Jacobson AL, et al. Using somatic mutations from tumors to classify variants in mismatch repair genes. Am J Hum Genet. 2018;103:19–29. Article  CAS  Google

Scholar  * Pagenstecher C, Wehner M, Friedl W, Rahner N, Aretz S, Friedrichs N, et al. Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants. Hum Genet. 2006;119:9–22.

Article  CAS  Google Scholar  * Brandão RD, Mensaert K, López-Perolio I, Tserpelis D, Xenakis M, Lattimore V, et al. Targeted RNA-seq successfully identifies normal and pathogenic splicing

events in breast/ovarian cancer susceptibility and Lynch syndrome genes. Int J Cancer. 2019;145:401–14. Article  Google Scholar  * Thakur PK, Rawal HC, Obuca M, Kaushik S. Bioinformatics

Approaches for Studying Alternative Splicing. In: Ranganathan S, Gribskov M, Nakai K, Schönbach, editors. Encyclopedia of Bioinformatics and Computational Biology (volume 2). Amsterdam,

Netherland: Elsevier B.V.; 2019, p. 221–34. * Tajaddod M, Tanzer A, Licht K, Wolfinger MT, Badelt S, Huber F, et al. Transcriptome-wide effects of inverted SINEs on gene expression and their

impact on RNA polymerase II activity. Genome Biol. 2016;17:220. Article  Google Scholar  * Katharina W, Tom C, Annekatrin W, Ludwine M. The NF1 gene contains hotspots for L1

endonuclease-dependent de novo insertion. PLos Genet. 2011;7:e1002371. Article  Google Scholar  * Qian Y, Mancini-DiNardo D, Judkins T, Cox HC, Brown K, Elias M, et al. Identification of

pathogenic retrotransposon insertions in cancer predisposition genes. Cancer Genet. 2017;216–217:159–69. Article  Google Scholar  * Wang L, Norris ET, Jordan IK. Human retrotransposon

insertion polymorphisms are associated with health and disease via gene regulatory phenotypes. Front Microbiol. 2017;8:1418. https://doi.org/10.3389/fmicb.2017.01418. Article  PubMed  PubMed

Central  Google Scholar  * Puurand T, Kukuškina V, Pajuste FD, Remm M. AluMine: alignment-free method for the discovery of polymorphic Alu element insertions. Mob DNA. 2019;10:31. Article 

Google Scholar  * Xing J, Zhang Y, Han K, Salem AH, Sen SK, Huff CD, et al. Mobile elements create structural variation: analysis of a complete human genome. Genome Res. 2009;19:1516–26.

Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Edanz (www.edanzediting.co.jp) for editing the English text of a draft of this manuscript. IM received generous

support from M. Kagawa, T. Ito, A. Yamamoto, and N. Kamae for providing clinical information. FUNDING This research was supported by Japan Agency for Medical Research and Development (AMED)

under grant JP 18kk0205004, JSPS KAKENHI Grant Number JP18K07339 and National Cancer Center Research and Development Found Grant Number 31-A-2. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Department of Molecular Diagnosis and Cancer Prevention, Saitama Cancer Center, 780 Komuro, Ina-machi, Kitaadachi-gun, Saitama, 362-0806, Japan Gou Yamamoto, Izumi Miyabe, Keisuke Tanaka, 

Miho Kakuta & Kiwamu Akagi * Department of Clinical Genetics, Juntendo University Graduate School of Medicine, Tokyo, Japan Motoko Watanabe * Department of Urology, Saitama Medical

Center, Saitama Medical University, Kawagoe, Saitama, Japan Satoru Kawakami * Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Kawagoe,

Saitama, Japan Hideyuki Ishida Authors * Gou Yamamoto View author publications You can also search for this author inPubMed Google Scholar * Izumi Miyabe View author publications You can

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ARTICLE Yamamoto, G., Miyabe, I., Tanaka, K. _et al._ SVA retrotransposon insertion in exon of MMR genes results in aberrant RNA splicing and causes Lynch syndrome. _Eur J Hum Genet_ 29,

680–686 (2021). https://doi.org/10.1038/s41431-020-00779-5 Download citation * Received: 06 April 2020 * Revised: 30 October 2020 * Accepted: 17 November 2020 * Published: 08 December 2020 *

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