CANCER GENOMICS & PROTEOMICS 18 : 531-542 (2021) doi:10.21873/cgp.20278 Several Fusion Genes Identified in a Spermatic Cord Leiomyoma With Rearrangements of Chromosome Arms 3p and 21q IOANNIS PANAGOPOULOS 1, LUDMILA GORUNOVA 1, KRISTIN ANDERSEN 1, INGVILD LOBMAIER 2 and SVERRE HEIM 1,3 1Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway; 2Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway; 3Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Abstract. Background/Aim: Benign smooth-muscle tumors, Leiomyomas are benign smooth -muscle tumors . They have leiomyomas, occur in nearly every organ but are most been described in nearly every organ but are most common common in the uterus. Whereas much is known about the in the uterus (fibroids) (1-5). genetics of uterine leiomyomas, little genetic information Much is known about the genetics , and hence the exists about leiomyomas of other organs. Here, we report pathogenesis, of uterine leiomyomas (6-9). In brief, most and discuss the genetic findings in a para-testicular uterine leiomyomas are cytogenetically characterized by the leiomyoma. Materials and Methods: Cytogenetic, array presence of one or more of the following cytogenetic comparative genomic hybridization (aCGH) RNA aberrations: t(12;14)(q15;q23–24) ; del(7)(q21.2q31.2) ; sequencing, reverse-transcription polymerase chain reaction rearrangements involving 6p21, 10q 22, and 1p ; trisomy 12 ; (RT- PCR), and Sanger sequencing analyses were performed deletions of 3q ; and changes of the X chromosome (10, 11). on a leiomyoma of the spermatic cord removed from a 61- These chromosomal aberrations rearrange and deregulate year-old man. Results: The karyotype was 48~50,XY,add(3) the following genes : High mobility group AT-hook 2 (p21),+4,+7,+8,+9,add(21)(q22)[cp9]/46,XY[2]. aCGH (HMGA2 ) on 12q14, RAD51 paralog B (RAD51B ) on confirmed the trisomies and also detected multiple gains and 14q24, cut -like homeobox 1 ( CUX1 ) on 7q22 , procollagen losses from 3p and 21q. RNA sequencing detected the C-endopeptidase enhancer ( PCOLCE ) on 7q22 , HMGA1 (on chimeras ARHGEF3–CACNA2D2, TRAK1–TIMP4, ITPR1– 6p21), lysine acetyltransferase 6B ( KAT6B ) on 10q22 , and DT-NR2C2, CLASP2–IL17RD, ZNF621–LARS2, CNTN4– collagen type IV alpha 5 chain/collagen type IV alpha 6 RHOA, and NR2C2–CFAP410. All chimeras were confirmed chain (COL4A 5/ COL 4A6 ) on Xq 22 (6-9). Additionally, by RT-PCR and Sanger sequencing. Conclusion: Our data, point mutations of the mediator complex subunit 12 together with those previously published, indicate that a (MED12 ) gene (on Xq13) have also been reported to be group of leiomyomas may be cytogenetically characterized frequent in uterine leiomyomas (12-15). Very little genetic by aberrations of 3p and the formation of fusion genes. information exists on leiomyomas of other organs , such as gastrointestinal (16-20) and retroperitoneal leiomyomas (21- 23). A single para-testicular leiomyoma has also been genetically examined (24). In the present study, we report rearrangements of the p This article is freely accessible online. arm of chromosome 3 in a leiomyoma of the spermatic cord leading to multiple fusion genes. We review the literature Correspondence to: Ioannis Panagopoulos, Section for Cancer and conclude that such 3p aberrations may be characteristic Cytogenetics, Institute for Cancer Genetics and Informatics, The of a leiomyoma subgroup. Norwegian Radium Hospital, Oslo University Hospital, Montebello, PO Box 4954 Nydalen, NO-0424 Oslo, Norway. Tel: +47 22782362, e-mail: [email protected] Materials and Methods Key Words: Spermatic cord leiomyoma, para-testicular leiomyoma, Ethics statement . The study was approved by the regional Ethics cytogenetics, chromosome arm 3p, fusion genes. Committee (Regional komité for medisinsk forskningsetikk Sør-Øst, 531 CANCER GENOMICS & PROTEOMICS 18 : 531-542 (2021) Norge, http://helseforskning.etikkom.no ) and written informed consent was obtained from the patient . The Ethics Committee’s approval included a review of the consent procedure. All patient information has been de-identified. Case description . A small lump was detected in the groin of a 60- year-old man. Core -needle biopsy showed a leiomyomatous tumor with uncertain malignant potential. The tumor was resected. The tumor consisted of bundles of smooth muscle cells without atypia (Figure 1) . There was no mitotic activity nor necrosis . The final diagnosis was spermatic cord leiomyoma. G-Banding karyotyping and array comparative genomic hybridization (aCGH) . analyses . Fresh tissue from a representative area of the tumor was analyzed cytogenetically as previously described (25). Genomic DNA was extracted from the tumor using the Maxwell RSC Instrument and Maxwell RSC Tissue DNA Kit (Promega, Madison, WI , USA) , the concentration was measured using Quantus Fluorometer and the QuantiFluor ONE dsDNA System (Promega) , and aCGH was performed using CytoSure array products (Oxford Gene Technology , Begbroke, Oxfordshire, UK ) as previously described (26). RNA sequencing. Total RNA was extracted from frozen tumor tissue adjacent to that used for cytogenetic analysis and histological examination using miRNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA from the tumor was sent to the Figure 1. Microscopic examination of the spermatic cord leiomyoma. Genomics Core Facility at the Norwegian Radium Hospital, Oslo Hematoxylin and eosin-stained section showing bundles of elongated University Hospital for high-throughput paired-end RNA- cells with cigar-shaped nuclei and eosinophilic cytoplasm. Original sequencing . Fusion transcripts were found using FusionCatcher magnification, A: 20×; B: 40×. software (27, 28). Reverse transcription PCR (RT -PCR ) and Sanger sequencing analyses. The primers used for PCR amplification and Sanger sequencing analyses are shown in Table I. The primer RNA sequencing analysis with FusionCatcher detected combinations and the corresponding fusion transcripts are seven fusion genes (Table III , Figure 3A). One transcript was presented in Table II. The iScript Advanced cDNA Synthesis Kit tris for RT-PCR was used to reverse transcribe 1 μg of total RNA detected for each of the chimeras inositol 1,4,5- phosphate according to the manufacturer’s instructions (Bio-Rad, Hercules, receptor divergent transcript –nuclear receptor subfamily 2 CA, USA). cDNA corresponding to 20 ng total RNA was used as group C member 2 (ITPR1–DT –NR2C2 ), cytoplasmic linker template in subsequent PCR assays. BigDye Direct Cycle associated protein 2 –interleukin 17 receptor D ( CLASP2– Sequencing Kit was used to perform both PCR and cycle (Sanger) IL17RD ), nuclear receptor subfamily 2 group C member 2 – sequencing according to the companyʼs recommendations cilia and flagella -associated protein 410 ( NR2C2–CFAP410 ), (ThermoFisher Scientific, Waltham, MA, USA). Sequencing was zinc finger protein 621 –leucyl -tRNA synthetase 2, run on an Applied Biosystems SeqStudio Genetic Analyzer ZNF621–LARS2 system (ThermoFisher Scientific). For computer analysis of mitochondrial ( ), and contactin 4 –ras sequence data, the basic local alignment search tool (BLAST) homolog family member A ( CNTN4–RHOA ), whereas two software was used (29). chimeric transcripts were detected for the fusion genes Rho guanine nucleotide exchange factor 3 –calcium voltage-gated Results channel auxiliary subunit alpha2delta 2 (ARHGEF3 – CACNA2D2 ) and trafficking kinesin protein 1–TIMP The G-banding analysis (Figure 2A) yielded the following metallopeptidase inhibitor 4 ( TRAK1 –TIMP4 ) ( Table III) . In karyotype : six of the fusion genes , both the 5 ’- end and 3 ’- end fusion 48~50,XY,add(3)(p21),+4,+7,+8,+9,add(21)(q22)[cp9]/46,XY[2]. partner genes were mapped on chromosome arm 3p (Table III, Figure 3A) . In the seventh, the NR2C2-CFAP410 aCGH confirmed trisomy for chromosomes 4,7,8, and 9 chimera , the 5 ’- end partner gene, NR2C2 , was from 3p25.1 , (Figure 2B) . In addition, multiple gains and losses were whereas CFAP410 was from 21 q22 .3 (Tables I and III) . All detected on the p arm of chromosome 3 (Figure 2C) and on fusion transcripts were verified using RT-PCR and Sanger the q arm of chromosome 21 (Figure 2D) . sequencing analyses (Figure 3B). 532 f ( f g C D g t b c a c s o i u 4 i t e a b t b a d l r l t d u 1 a c t l i i o a t n 4 e a p P D n e h n h e e o m ( u o n r N u e h h o h x d r b n f t l 6 t e f 2 e l e b e e y a t 2 a r 1 N o i T i i v b e e e a f i m g m e g v s i o r s n e t r i n n n d e d r , o ) o o s R s q T R T m b s 1 e 6 2 o m r d X s o o i r u o – i a e e c . 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