Detection of a complex chromosomal rearrangement in a novel mouse mutant by optical genome mapping

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Wight This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9271724/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Here we highlight the utilities of optical genome mapping (OGM) in determining the genomic rearrangements present in a novel transgenic mouse line (Line 781), which expresses the bacterial lacZ reporter gene under control of the mouse myelin proteolipid protein ( Plp1 ) promoter. Hemizygous transgenic mice from Line 781 present with a mutant phenotype (documented here) which entails small body size and paws and craniofacial aberrations that are 100% penetrant, whereas their non-transgenic littermates are phenotypically normal. OGM was used to determine that the transgene sits at the intersection of an unbalanced reciprocal translocation between chromosomes 1 and 2, with deletion of approximately 3.9 (chr1) and 1.8 (chr2) Mbp from the rearranged (derivative) chromosomes, thus resulting in a monosomy over these regions in the mutant genome. As well, OGM was able to determine the number of full-length copies of transgene that integrated and their orientation. Sanger sequencing of PCR products that span a junction were used to determine the chromosomal breakpoints and transgene integration site, precisely. The complex chromosomal rearrangements in Line 781 span 38 protein-coding genes that result in the transection of 1 gene from chr1 and deletion of 33 and 4 genes from chr1 and chr2, respectively. The resulting mutant phenotype is consistent with 1q24 deletion syndrome in humans having an interstitial deletion of the syntenic region in Chr1. Thus, our mouse mutant may serve as an animal model, in future studies, to explore the molecular and cellular basis of anomalies present in patients with 1q24 deletion syndrome. Optical genome mapping Unbalanced reciprocal translocation Structural variant Mouse mutant FISH analysis Transgenic mouse Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Genomic rearrangements constitute large-scale structural alterations in DNA ranging in size from just over 50 bp to megabase pairs as the result of duplications, deletions, insertions, inversions, or translocations (for reviews see: Carvalho and Lupski 2016 ; Collins and Talkowski 2025 ; Ho et al. 2020 ; Laufer et al. 2023 ). While not all constitutional (germline) structural variants (SVs) lead to aberrant phenotypes or health-related issues, a fair number of them give rise to genomic disorders due to gene disruption/dysregulation, copy number variation (CNV) of a dosage-sensitive gene or non-coding cis -regulatory element, or generation of a fusion gene whose product has altered activity or function (Harel and Lupski 2018 ; Hu et al. 2018 ; Spielmann and Klopocki 2013 ; Stankiewicz and Lupski 2010 ; Weischenfeldt et al. 2013 ). A variety of methods exist for detection of novel SVs, from low resolution cytogenetic techniques such as fluorescent in situ hybridization (FISH) to high resolution techniques such as long-read sequencing, whose utilities are often subject to the type of SV under investigation (De Coster et al. 2019; Ho et al. 2020 ; Moustakli et al. 2025 ; Porubsky and Eichler 2024 ; Yang 2020 ). One of the newest techniques to be used for discovery of SVs is optical genome mapping (OGM) (Chan et al. 2018 ; Garcia-Heras 2021 ; Mantere et al. 2021 ; Qu et al. 2023 ; Zhang et al. 2025 ). The technology consists of adding an optical/fluorescent tag to a particular target site throughout the genome and measuring the distance between successive tags in each molecule of ultra-high molecular weight DNA (Dremsek et al. 2021 ; Levy et al. 2025 ). The reads are then de novo assembled locally and compared to a reference genome. OGM has been used to identify balanced and unbalanced reciprocal translocations (RTs) (Lederbogen et al. 2024 ; Montjean et al. 2025 ; Ogiwara et al. 2022 ; Schnause et al. 2021 ; Zhang et al. 2023 ) among other types of SVs, and even aid in localizing a transgene integration site in mouse (Ding et al. 2023 ). The current study highlights some of the unique attributes of OGM amid discovery of a complex SV marked by a transgene in a mouse line. The transgene uses the mouse myelin proteolipid protein ( Plp1 ) promoter to drive expression of a bacterial lacZ expression cassette (Patyal et al. 2020 ). The lacZ gene has been used widely as a reporter in transgenic mice due to the innocuous nature of its gene product (β-galactosidase) in mammalian cells (Cui et al. 1994 ). Our group has generated over twenty transgenic mouse lines using the same lacZ expression cassette without any untoward consequences except in one, Line 781 (Hamdan et al. 2018 ; Patyal et al. 2020 , 2023 ; Wight et al. 1993 ). While correct spatiotemporal expression of the Plp1-lacZ transgene remains intact in Line 781 (Patyal et al. 2020 ), mice hemizygous for the transgene have small body size and paws and dysmorphic facial features compared with their nontransgenic littermates. The mutant phenotype is 100% penetrant with the transgene. Initially, we surmised that integration of the transgene may have disrupted a critical gene in Line 781. However, FISH analysis presented in this study indicates that the chromosome which harbors the transgene also has a nearby reciprocal translocation (RT), which alternatively could trigger the aberrant phenotype. A RT is said to be balanced if there is no net gain or loss of genetic material (Wilch and Morton 2018 ). Typically, balanced RTs do not result in an overt phenotype in the carrier unless the translocation dissociates a critical distal regulatory element from a gene sensitive to variations in copy number or transects the gene body. Instead, phenotypic abnormalities are more likely to arise from unbalanced RTs due to deletion (De Gregori et al. 2007 ) or duplication (Howarth et al. 2011 ) of genetic material at the breakpoint junction. In the current study, we used OGM to discern the type of RT present in mutant animals from Line 781. The data revealed that the RT in this line is unbalanced, resulting in the deletion of more than 30 genes from the affected chromosomes combined. Besides determining the approximate location of deleted sequence in the derivative (der) chromosomes, OGM determined that the transgene integrated within a single site of the genome at or close to the breakpoint junction in der2. Due to asymmetric labeling of the transgene sequence by OGM, we were able to determine the number of full copies that integrated, as well as their orientation. Sanger sequencing of PCR products that traverse the RT breakpoints in der2 established that the transgene copies lay directly between the intersection of chromosomal pieces. Thus, it is likely that the underlying etiology in mutant animals from Line 781 may be due to the loss of one or more dosage-sensitive genes from the derivative chromosome(s). Materials and Methods Animals Mice from Line 781 were used in the study. Generation of mice which harbor a lacZ transgene [mPLP(+)Z/FL] driven by the mouse Plp1 promoter has been described, previously (Patyal et al. 2020 ). All procedures involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences (UAMS) in compliance with the US Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. Mice were produced by breeding hemizygous transgenic (i.e., mutant) males with C57BL/6 wild-type females. The resulting progeny was genotyped for presence of the transgene by PCR analysis with genomic DNA isolated from tail biopsies according to the methods of Truett et al. ( 2000 ) and the lacZ primer pair described by Stratman et al. ( 2003 ). Because the aberrant phenotype in Line 781 is 100% penetrant with the transgene, mice will be distinguished by phenotype—mutant or wild-type—instead of genotype, henceforth. Micro-CT analysis Male mice at 6 months of age were euthanized by isoflurane overdose followed by cervical dislocation to ensure death. Heads were harvested, skin removed, and subsequently fixed in 10% neutral buffered formalin at 4°C for 24h, washed 3 times (1 min, each) in phosphate buffered saline (pH 7.4), and stored long-term in 70% ethanol. Micro-CT images were acquired using the micro-CT 45 machine from SCANCO Medical AG (Brüttisellen, Switzerland). Each sample was placed in a 25.2 mm diameter tube and scanned at 55 kV, 72 µA and 4W using a 0.5mm Al filter. A medium resolution scan (voxel size = 24.5 µm) of the entire skull was obtained. Coronal and sagittal images were rendered using the RayTracer (SCANCO) software, which generates a 3D reconstruction of the scanned skull. Longitudinal growth study Body weight and length was measured weekly in a cohort of mutant and wild-type mice from Line 781 between 3–16 weeks of age. Mice were lightly anesthetized with 2% isoflurane in a chamber prior to measurement. Body length indicates the distance (cm) from nose to anus. Comparisons were made between mutant and wild-type mice of the same sex. Data are presented as the mean ± standard deviation (SD) at each age for a specific phenotype (genotype). Results were considered statistically significant if the p-value was less than 0.05 using a standard t-test. FISH analysis Lymphocytes were isolated from the spleen of a mutant mouse from Line 781 at SeeDNA Biotech Inc. (Windsor, Ontario, Canada). The cells were cultured at 37°C in RMPI 1640 medium supplemented with fetal calf serum, concanavalin A, lipopolysaccharide and mercaptoethanol. Subsequently, cells were harvested and chromosomal slides prepared by conventional methods (hypotonic treatment, fixation, and air dry) employed at SeeDNA. Initially, the mPLP(+)Z/FL transgene probe was labeled with digoxin and detected by rhodamine. The procedure for hybridization and detection was performed according to published methods (Heng and Tsui 1993 ; Heng et al. 1992 ). FISH signals were observed under fluorescent microscopy. Images were captured with a charged-coupled device (CCD) camera and merged using RS Image software. Additional FISH analysis was performed likewise, except the transgene probe was labeled with biotin and detected by fluorescein isothiocyanate (Heng et al. 1992 ), in conjunction with mouse chromosome 1 paint probes (Cambio, Cambridge, UK) labeled with Cy3. Optical genome mapping (OGM) OGM sample preparation, mapping, and bioinformatics analysis was conducted by the Molecular Genomics core at Texas A&M University (College Station, TX, USA). Ultra-high molecular weight DNA isolated from liver of a mutant female mouse (Line 781) was labeled with DL-green fluorophores at CTTAAG sites with DLE-1 methyltransferase. The label density averaged 15.017 labels per 100 kb. The average length of molecules (DNA fragments) was 246.092 kbp. The contig N50 was 243.616 kbp. The de novo assembly pipeline in the Bionano Solve software (version 1.6.1) was used to build the mutant mouse genome from Line 781, with GRCm38.p6 as the reference genome. The rare variant analysis pipeline in the Bionano software was used to map the translocation breakpoints, associated deletions, and transgene integration site/copy number, upon consideration of the transgene sequence (Patyal et al. 2020 ), which contains several target sites for labeling. The confidence cutoff was 0.95. PCR and sequencing validation of breakpoints and transgene integration site PCR analysis was performed using primers that tile the chromosomal regions in the vicinity of the breakpoints predicted by OGM. Sequences of the primers, as well as a couple of transgene-specific primers, are provided in Table 1 . The transgene-specific primers (Tg-1 and Tg-2) were fixed (invariable) in the PCR reactions for amplification of products from der2. Chr2-specific sense primers der2-2A to der2-2F were used separately, in conjunction with Tg-2 as the antisense primer. Alternatively, chr1-specific antisense primers der2-1A to der2-1F were used individually, along with the Tg-1 sense primer. To amplify sequences that traverse the RT junction in der1, a chr1-specific sense primer (der1-1A to der1-1F) was paired with a chr2-specific antisense primer (der1-2A to der1-2F). Genomic DNA isolated from the liver of a female mutant mouse (Line 781) was used as a template in the reactions. PCR reactions (25 µL, total) consisted of 50 ng of genomic DNA, primers each at a final concentration of 0.5 µM final, and Platinum SuperFi Master Mix (Invitrogen, Waltham, MA, USA) according to the manufacturer’s specifications. PCR conditions were as follows: 30 sec at 95°C (denaturation), 20 sec at 65°C (annealing), and 60 sec at 72°C (extension) for 35 cycles. The PCR products were resolved by gel electrophoresis on a 0.7% agarose gel. PCR was repeated again for those primer pairs that yielded the smallest product across each of the three junctions; der2 has two junctions since the transgene integrated directly at the intersection of the RT. Serial PCR reactions were performed similarly, except that 1.25 µL of the reaction mixture from the first round of PCR was used as template DNA. Products from the serial round of PCR were resolved on a 0.7% agarose gel, bands excised, and DNA purified using the QIAquick Gel Extraction Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s recommendations. DNA sequences of the PCR products were determined using the primers listed in Table 2 , which were named according to the primer source (chr1, chr2, Tg) followed by the target chromosome (der1, der2). Sanger sequencing was performed at GENEWIZ (Azenta Life Sciences, South Plainfield, NJ, USA) and the DNA Sequencing Core Facility at UAMS. Table 1 PCR primers* *Tg-2 serves as an antisense primer for der2-2 sense primers; Tg-1 serves as a sense primer for der2-1 antisense primers Primer Sequence Positions (GRCm38.p6) der2-2A 5′-GAGAGGCTTATTGGACATCTAAAATATCG-3′ chr2:106476542–106476570 der2-2B 5′-CTTAGATCTTCCTTTTATTCGCTGAGAAG-3′ chr2:106478379–106478407 der2-2C 5′-GAATAGTTCCTTCTAATTTCTCCCTCTACG-3′ chr2:106478843–106478872 der2-2D 5′-GCTATAGCTAGCAAATATTCTCCAATCAG-3′ chr2:106479200–106479228 der2-2E 5′-TCATCACTTCTCTATGTCCTGGTGTTTTC-3′ chr2:106479256–106479284 der2-2F 5′-TGCCACCTCTGTGTAAATGACTAAATGT-3′ chr2:106479603–106479630 Tg-2 5′-CCGCTATTTCTCTGTTCTCGCTATTATTC-3′ der2-1A 5′-GGTATATCCTATTTTCTCCTTAAGTGCCTC-3′ chr1:163466837–163466808 der2-1B 5′-CTTGAGTTTAGCCAAACAAACTATGGTTAC-3′ chr1:163466310–163466281 der2-1C 5′-GAAAGACGGTGAAATACAGTAAGTAGGAAG-3′ chr1:163466164–163466135 der2-1D 5′-ATCAAATCAAATTCAGACAGAGACCTGTAG-3′ chr1:163465279–163465250 der2-1E 5′-GTTAGTTAGACAGTGATTTCCCCAAAGATG-3′ chr1:163464271–163464242 der2-1F 5′-AAGACGGAGGTCAATAAGATGGATATAAG-3′ chr1:163463780–163463752 Tg-1 5′-CTACCTGTGTCTATGATTCTGCTTTCATTG-3′ der1-1A 5′-GTCTTATATTATCAAAGGACTGCTTCCC-3′ chr1:159563576–159563603 der1-1B 5′-CTGAGAATTATGGGTGGATATTTTGGATTG-3′ chr1:159565280–159565309 der1-1C 5′-TAATTTTCCTGCACTCACTTACCAAGG-3′ chr1:159565724–159565750 der1-1D 5′-ATAACTAAGGCACAGGACTGATTGATG-3′ chr1:159566159–159566185 der1-1E 5′-CAATATGGTATCTATGAAAGCCTGTTTCCC-3′ chr1:159567660–159567689 der1-1F 5′-CACTCTTTCCTCAAAGGTTATTTTCTAGGG-3′ chr1:159568646–159568675 der1-2A 5′-GGATATTTTCTGAGCACCTTCTTTTTCTCC-3′ chr2:108257618–108257589 der1-2B 5′-CATTGTTATTGGTGTGTATGCTCTGATG-3′ chr2:108257149–108257122 der1-2C 5′-AGATTATTACCAAGAGCACTCCCTGTC-3′ chr2:108256829–108256803 der1-2D 5′-TTGAACTTTTGATTCTACTGTCTCCAG-3′ chr2:108255484–108255458 der1-2E 5′-CTTACTTTCTATTATTCCCACCACCTCC-3′ chr2:108255169–108255142 der1-2F 5′-GAGCAAACACCTATTTACCACAGATATTAC-3′ chr2:108254391–108254362 Table 2 DNA sequencing primers* *Tg-2 primers are set in the bottom strand of DNA; Tg-1 primers are set in the top strand of DNA Primer Sequence Positions (GRCm38.p6) chr2A-der2 5′-GCACTGGCATTTTAGAAGG-3′ chr2:106479090–106479108 chr2B-der2 5′-TCCATGAAACTACTCAAGTGTG-3′ chr2:106479125–106479146 Tg-2A 5′-TGTCCAAACTCATCAATGTATCTT-3′ Tg-2B 5′-AGGATTCAAGAACCCCTC-3′ Tg-2C 5′-CATAAAGAGAAGATGGAGCCC-3′ chr1A-der2 5′-ATTGTGGGGACCTACTTGCTG-3′ chr1:163465165–163465145 chr1B-der2 5′-AGCCACCACAACATGCAA-3′ chr1:163465074–163465057 Tg-1A 5′-CCAGTAAACAGTGTGCTTCATGC-3′ Tg-1B 5′-AGGATCAGGAGAGTCAGTG-3′ Tg-1C 5′-GCTGGGAAATGACAGAATGCC-3′ chr1A-der1 5′-TTTTCCTGCACTCACTTACC-3′ chr1:159565727–159565746 chr2A-der1 5′-TTGATTCTACTGTCTCCAGCC-3′ chr2:108255476–108255456 Results Transgenic mice from Line 781 display a mutant phenotype While generating mice that contain a Plp1-lacZ transgene one of the founders displayed craniofacial abnormalities and was smaller in size. Despite this, the founder was fertile and mouse Line 781 was established from him (Patyal et al. 2020 ). Interestingly, the mutant phenotype is 100% penetrant with the transgene. Therefore, we have elected to differentiate the mice based on phenotype (mutant or wild-type) instead of genotype. Figure 1 shows that mutant animals at 6 months of age exhibit smaller body length and paws, and craniofacial aberrations including more prominent cranial sutures, smaller orbital fossae, and a compressed snout that is less pointed compared with wild-type mice. To quantitate the diminished size of the mutant longitudinally, body weight and length (nose to anus) was measured weekly in a cohort of mutant and wild-type mice from Line 781 between 3–16 weeks of age. As shown in Fig. 2 , both weight and length of the mutants were significantly decreased compared with wild-type (same sex) counterparts, at all ages examined. Thus, the small size of the mutant originates early in development. FISH analysis indicates mutant mice from Line 781 have a RT Initially, we suspected that insertion of the transgene must have disrupted a critical gene. Hence, FISH analysis was performed with lymphocytes isolated from the spleen of a mutant mouse to determine the chromosomal location of the transgene. As illustrated in Fig. 3 , the transgene integrated in chr2-specific sequence, but this chromosome—der2—also has a RT with chr1, close to the transgene integration site. This explains the reduction in litter size (~ 3 mice/litter; data not shown) by ~ 50% when a mutant animal (hemizygous for the transgene) is mated with a wild-type mouse, due to unbalanced gametes in the carrier of the RT (Rodríguez et al. 2010 ). Thus, the mutant phenotype is likely due to the RT itself. OGM reveals that the RT in Line 781 is unbalanced To better assess the RT breakpoints and location of the transgene in Line 781, OGM was performed with ultra-high molecular weight genomic DNA obtained from a mutant female mouse, with an average length of 246.092 kbp. The DNA was labeled with DL-green fluorophores at CTTAAG sites with DLE-1 methyltransferase. Because the target site is palindromic, both DNA strands get labeled. The label density averaged 15.017 labels per 100 kb. The labeled DNA was analyzed on the Bionano Saphyr® system to measure the distance between labels (tags) for each single, extremely long strand of genomic DNA (‘molecule’). The resulting data was entered into the de novo assembly pipeline to group the molecules by label pattern similarity in order to create maps, which then were used to construct the mutant genome. The rare variant analysis pipeline was used to compare the assembly against a reference genome (GRCm38.p6). Areas of discrepancy within the mutant genome were identified using the structural variant (SV) caller. The SV caller indicated that the RT junction in der2 was separated by extraneous DNA. Based on the array of target sites for labeling within the transgene sequence, the extraneous DNA was determined to be the transgene. It is estimated that five complete copies of the transgene integrated in a tail-to-head orientation, directly between the breakpoints in der2. As shown in Fig. 4 , the breakpoints in der2 were estimated to be in the vicinity of positions chr2:106,476,853 and chr1:163,466,814. The SV caller also estimated the RT junction in der1 occurs near positions chr1:159,566,643 and chr2:108,255,381. Taken together, the data suggest that parts of chromosomes 1 and 2 were deleted during the reorganization of the mutant genome. Copy number variant (CNV) analysis was performed for the mutant genome using the Bionano software. CNV analysis quantitates the depth of coverage of a particular region, relative to the expected depth of the entire genome (Sahajpal et al. 2021 ). The analysis indicated areas of monosomy within chromosomes 1 and 2 (Fig. 5 ), which correspond to gaps of missing sequence from the derivative chromosomes when the SV caller estimated breakpoints are considered in combination. Thus, the RT is unbalanced, with segments of chr1 and chr2 missing from the derivative chromosomes. Identification of the RT breakpoints in Line 781 Sanger sequencing of PCR products which traverse junctions between breakpoints (including with the transgene) was used to determine the precise location of chromosomal rearrangements in mutant mice from Line 781. Primers were designed that tile chr1- and chr2-specific regions of the derivative chromosomes near the breakpoints estimated by the SV caller, which reflect the last labeled site that aligns with the reference chromosome prior to the breakpoint. The primers (Table 1 ) were named according to the derivative chromosome they target (der1 or der2), followed by the chromosome number the sequence was derived from (1 or 2), and a letter (A–F) to distinguish the individual primers within a given tiled region (‘A’ indicates the 5′-most primer on a particular strand). Because the RT breakpoints in der2 are separated by copies of the transgene, transgene-specific primers (Tg-1 and Tg-2) were also generated and used as fixed primers in the PCR reactions for der2. The transgene-specific primers were numbered according to the chromosome targeted and used jointly in PCR reactions to amplify sequences across the junction of full-length copies of the transgene integrated tail-to-head, producing a product of ~ 456 bp (data not shown). Primer pairs der2-2C in conjunction with Tg-2, and der2-1D in conjunction with Tg-1, generated the shortest PCR products of the tiled primers for der2, while der1-1C together with der1-2D resulted in the smallest size product for der1 (data not shown). Use of the upstream tiled primers resulted in larger size products consistent with their chromosomal location, validating that the amplicons span a breakpoint. The downstream tiled primers did not produce a product, implying that the sequence is absent from the derivative chromosome. Primer pairs that generated the smallest PCR product across each of the junctions in the mutant genome were gel purified and sequenced using the primers listed in Table 2 . Due to the relatively large size of amplicons generated from der2, multiple primers were needed to fully sequence a strand. The results determined that the transgene integrated between chr2:106,479,179 and chr1:163,464,519 in der2 (Fig. 6 ). Interestingly, partial copies of the transgene are present at both ends of the transgene integration site. Moreover, there is an insertion of 21 bp of inverted chr2 sequence (positions 108,254,948–108,254,928) located directly between the transgene and chr1 sequence in der2. The junction for der1 is situated between chr1:159,565,980 and chr2:108,255,242 (Fig. 6 ), directly separated by a 76-bp insertion of inverted chr2 sequence (positions 108,255,235–108,255,160). Taken together, these results indicate that portions of chr1 (159,565,981–163,464,518) and chr2 (106,479,180–108,255,241 except for insertion of inverted 108,255,235–108,255,160) are missing from the derivative chromosomes in the mutant. Table 3 shows the protein-coding genes that lay within these deleted regions, encompassing 34 genes in chr1 and 4 genes in chr2. Because the breakpoint occurs within intron 1 of Tnr , only the proximal portion of the gene remains in der1. The rest of the genes are completely missing from the derivative chromosomes. Table 3 Protein-coding genes deleted in der1/der2 Gene Positions (GRCm38.p6) Tnr chr1:159523729–159931729 Tnn chr1:160085029–160153692 Mrps14 chr1:160195215–160201186 Cacybp chr1:160202367–160212892 Rabgap1l chr1:160219174–160793476 Gpr52 chr1:160574717–160579704 Rc3h1 chr1:160906411–160974976 Serpinc1 chr1:160978583–161002543 Zbtb37 chr1:161002922–161034864 Gas5 chr1:161034601–161038539 Dars2 chr1:161037580–161070668 Cenpl chr1:161070767–161086724 Klhl20 chr1:161088375–161131508 Ankrd45 chr1:161142447–161170510 Tex50 chr1:161156843–161159314 Prdx6 chr1:161240112–161251210 Tnfsf4 chr1:161395438–161418206 Tnfsf18 chr1:161494657–161505290 Fasl chr1:161780691–161788495 Suco chr1:161816112–161876837 Pigc chr1:161969188–161973460 Dnm3 chr1:161982450–162478321 Mettl13 chr1:162533671–162548541 Vamp4 chr1:162570515–162599082 Myoc chr1:162639150–162649694 Prrc2c chr1:162671785–162740560 Fmo4 chr1:162793188–162816072 Fmo1 chr1:162829561–162866610 Fmo2 chr1:162874317–162898758 Fmo6 chr1:162916551–162937566 Fmo3 chr1:162953799–162984528 Mroh9 chr1:163024302–163085670 Prrx1 chr1:163245119–163315145 Gorab chr1:163384903–163403669 Mpped2 chr2:106693181–106868361 Arl14ep chr2:106962529–106974397 Fshb chr2:107055986–107059651 Kcna4 chr2:107290589–107326804 Discussion Here we report that mutant mice from Line 781 have an unbalanced RT with 5 full-length copies of a Plp1-lacZ transgene inserted between the translocation junction in der2. Thus far, only a few cases of transgenes integrating at or near a RT breakpoint have been reported (Durkin et al. 2001 ; Francke et al. 1992 ; Kasai et al. 2007 ; Mahon et al. 1988 ). This may be an underrepresentation due to investigators opting not to generate transgenic lines from founders with unexpected phenotypes, or to embryonic lethality. In the case of Line 781, the mutant phenotype (small body size and paws, craniofacial abnormalities) does not obfuscate spatiotemporal expression of the transgene (Patyal et al. 2020 ). Initially, we surmised that integration of the transgene in Line 781 disrupted a critical gene (or genes), which results in the mutant phenotype. However, FISH analysis established that an RT between chromosomes 1 and 2 exists in the mutant genome (Fig. 3 ), which likely elicits the phenotype. Ligation-mediated PCR (Rosenthal and Jones 1990 ) was undertaken to attempt to walk out from the transgene into the flanking chromosomal regions, but only junctions between adjoining copies of transgene were detected (data not shown). Therefore, OGM was employed to narrow down the junctions between different chromosomal regions or with the transgene. While OGM cannot identify chromosomal rearrangements to the nucleotide level, it has much better resolution than FISH (Levy et al. 2025 ). Moreover, CNV analysis of the OGM data was key in establishing that the RT in Line 781 is unbalanced (Fig. 5 ). Knowledge gained from the results with OGM was utilized to design primers that tile chromosomal regions surrounding the estimated breakpoints in the mutant genome for use in long-range PCR, at times in conjunction with fixed primers containing transgene sequence. Sanger sequencing of gel-extracted PCR products determined that the actual breakpoints were in fairly close proximity to those predicted by OGM; 663 and 139 bp apart in der1 for chr1 and chr2 segments, respectively, and 2,326 and 2,295 bp apart in der2 for chr2 and chr1 segments, respectively. Sanger sequencing also determined that four other breaks occurred within chr2, which resulted in small portions of inverted chr2 sequence being inserted at the translocation junction and transgene integration site of der1 (76 bp) and der2 (21 bp), respectively (Fig. 6 ). These rearrangements, coupled with insertion of the transgene at the translocation junction of der2, suggest that a series of recombinational events took place during microinjection of the transgene. Perhaps integration of the transgene caused chromothripis of portions of chr1 and chr2 during the repair process. The rearrangements are very stable since the mutant phenotype of the (Line 781) founder has been passed down for many generations. To the best of our knowledge, only one other study (Ding et al. 2023 ) has used OGM to map the location of a transgene in mouse, thus far. It was possible to determine that five full-length copies of the transgene integrated in a tail-to-head orientation in der2 by OGM (Fig. 4 ) based upon the array of target sites within the transgene sequence, which result in a distinct labeling pattern. Because the RT in Line 781 is unbalanced with deletion of approximately 3.9 (chr1) and 1.8 (chr2) Mbp of sequence in the derivative chromosomes, a monosomy exists over these regions in the mutant genome (Fig. 5 ). All total; 38 protein-coding genes are located within the deleted sequence (Table 3 ). It is conceivable that the mutant phenotype arises from a haploinsufficiency due to an insufficient amount of gene product from one (or more) of these genes needed to preserve its function (Johnson et al. 2019 ; Veitia et al. 2025 ). Intriguingly, microdeletion of portions of the chr1 syntenic region have been reported in patients with 1q24 microdeletion syndrome that present with growth deficiency (weight and height), small hands and feet, and dysmorphic facial features (Ashraf et al. 2015 ; Burkardt et al. 2011 ; Lefroy et al. 2018 ; Nishimura et al. 2010 ; Shepherdson et al. 2021 ; Yu et al. 2021 ). The condition is inherited in an autosomal dominant fashion (Lefroy et al. 2018 ; Shepherdson et al. 2021 ; Yu et al. 2021 ), consistent with mutant mice from Line 781. Several reports (Ashraf et al. 2015 ; Lefroy et al. 2018 ; Shepherdson et al. 2021 ; Yu et al. 2021 ) have ascribed the anomalies in patients with 1q24 deletion syndrome to a monosomy of the noncoding RNA gene, DNM3OS , which is located on the opposite strand of DMN3 intron 14. However, while Dnm3os knockout mice display skeletal and craniofacial deformities, heterozygous mice with one functional allele were described as phenotypically normal according to the authors (Watanabe et al. 2008 ), although the weight of heterozygotes appeared to trend lower than wild-type mice at 5 months of age (but not at P30) and there appeared to be some shortening of the sagittal axis of the skull in heterozygotes at P60. Thus, deletion of one allele of Dnm3os cannot fully explain the mutant phenotype in Line 781. Interstitial microdeletion of the chr2 syntenic region in humans (11p14.1), in contrast, is associated with ADHD, autism, developmental delay, and obesity (Shinawi et al. 2011 ). Hence, it is likely that the growth deficiency as well as other aberrations observed in mutant animals from Line 781 are associated with loss of a gene or genes from the affected locus of chr1. Conclusions This study highlights some of the unique attributes of OGM amid discovery of a complex SV marked by a transgene in mouse Line 781, which causes an aberrant phenotype in mice that harbor the transgene. With OGM we were able to determine the genomic rearrangements present in the mutant genome, which consist of an unbalanced RT between chr1 and chr2 and a corresponding loss of 3.9 (chr1-specific) and 1.8 (chr2-specific) Mbp of sequence from the derivative chromosomes. Furthermore, OGM was able to determine that five full-length copies of transgene integrated directly between the breakpoint junction of der2 in a tail-to-head orientation. In general, OGM can be used to distinguish the type of RT (balanced vs . unbalanced) and the identity of chromosomes involved. In addition, OGM can be used to determine whether a transgene integrated at a single or multiple site(s) in the genome and into which chromosome, as well as the number of full-length copies of transgene that integrated and their orientation provided that the transgene sequence contains an asymmetric distribution of target sites for labeling. Because a variety of enzymes with different targets are available for OGM, the choice of enzyme should center on the transgene sequence under investigation. With respect to the specific genetic rearrangements observed in our mouse mutant, the resulting phenotype is consistent with 1q24 deletion syndrome in humans having an interstitial deletion of the syntenic region from a single autosome of Chr1. Thus, mutant mice from Line 781 may serve as an animal model, in future studies, to explore the molecular and cellular basis of small stature and other anomalies present in patients with 1q24 deletion syndrome. Declarations Author contributions GH and PAW conceptualization. GH data curation, formal analysis, and validation. GH and PAW investigation and visualization. PAW funding acquisition. GH wrote the first draft. PAW reviewed and edited the final revision. All authors discussed the content and approved the final version of the manuscript. Funding This research was supported in part by a Barton Pilot Study award from the College of Medicine at the University of Arkansas for Medical Sciences and an R01 grant (NS106179) from the US National Institute of Neurological Disorders and Stroke. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Data Availability The OGM dataset is available at Mendeley Data, V2, doi: 10.17632/3h325jhj93.2. https://data.mendeley.com/datasets/3h325jhj93/2 Competing interests The authors declare no competing interests. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 04 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers invited by journal 01 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 30 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9271724","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617567487,"identity":"aa4b06d9-8605-4365-ae8e-88d3c0949927","order_by":0,"name":"Gretchen Holtgrefe","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Gretchen","middleName":"","lastName":"Holtgrefe","suffix":""},{"id":617567488,"identity":"538500eb-bbb8-45cc-9e09-3d6b4cb37bc8","order_by":1,"name":"Patricia A. Wight","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYFACxgZmBgYbKIeNaC0JaSRpYWAAajlMghZzieTm14U/zif2Tzv+gOFD2WGCOhgsZyS2Wc9IuJ0443aOAeOMc0RoMbid2GbMA9TScDuHgZm3jXgt5xLn305/wPyXSC3Nj3kSDiRuuJ1gwMxIjBbL+Q/bmHnSko03Av1ysOdcOmEt5jzHH3/msbGTnXc7/eGDH2XWRDgMGBcSMM4BwuohWpg/EKVyFIyCUTAKRi4AAAA2P8ErKrqbAAAAAElFTkSuQmCC","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Patricia","middleName":"A.","lastName":"Wight","suffix":""}],"badges":[],"createdAt":"2026-03-30 20:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9271724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9271724/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106311043,"identity":"8a647dd3-d6f8-481e-b204-8b3031e8b2b9","added_by":"auto","created_at":"2026-04-07 10:27:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":370502,"visible":true,"origin":"","legend":"\u003cp\u003eMice from Line 781 that are hemizygous for a \u003cem\u003ePlp1-lacZ\u003c/em\u003e transgene display a mutant (Mut) phenotype, whereas their non-transgenic littermates are phenotypically normal (i.e., wild-type; WT). Shown on the left side is an image of male mice taken at 6 months of age. Notice the body length and paws are smaller in the mutant and the snout is rounder compared to the WT littermate. Micro-computed tomography images of skulls from male mice at 6 months of age are shown on the right side and indicate that the cranial sutures in mutant mice are more prominent, the orbital fossa is smaller, and the snout is shortened and less pointed compared with WT mice.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/480290fd3b8b7016a1463df3.png"},{"id":106311028,"identity":"c216bb63-aaaf-4092-a4dc-0b05fb0f990d","added_by":"auto","created_at":"2026-04-07 10:27:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30821,"visible":true,"origin":"","legend":"\u003cp\u003eA longitudinal study of mutant and wild-type mice from Line 781 indicate that mutant mice are smaller in weight and body length (nose to anus) than wild-type (same sex) mice between 3 and 16 weeks of age. Plotted are the weekly mean ± SD for weight (top graphs) and body length (bottom graphs) with for wild-type mice denoted by filled symbols and those for mutants by open symbols. Results with males (n = 3 each) are shown in square symbols, while circles indicate females (n = 6 wild-type; n = 5 mutant). Significant differences were detected between wild-type and mutant animals of the same sex at all ages examined (p ≤ 0.035 males; p ≤ 0.015 females) using a standard t-test.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/420ea7f04bcc438e7a488d0c.png"},{"id":106311100,"identity":"3f887457-e91e-4222-ad3b-f0f376c506e5","added_by":"auto","created_at":"2026-04-07 10:28:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4487,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the genomic rearrangements in Line 781 by FISH analysis. The cartoon depicts the pertinent chromosomes in a mutant mouse from Line 781. FISH analysis revealed that the \u003cem\u003ePlp1-lacZ\u003c/em\u003e transgene (represented by purple circles) integrated in chr2 (in actuality, der2), which also had a RT with chr1. The circle atop each chromosome indicates the relative position of the centromere for the standard and derivative (der) chromosomes, illustrated. Chr1 sequence is portrayed in gray, while chr2 sequence is depicted in white.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/5b5910da1772939669f0ef9a.png"},{"id":106311099,"identity":"427865ee-eec5-4a85-9382-f12527b6c645","added_by":"auto","created_at":"2026-04-07 10:28:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":354349,"visible":true,"origin":"","legend":"\u003cp\u003eOptical genome mapping of the derivative chromosomes in a mutant mouse from Line 781. Using the structural variant caller with the OGM data estimated that the translocation junction for der1 lies near positions chr1:159,566,643 and chr2:108,255,381, whereas in der2, the transgene (indicated in purple) directly separates the translocation junction estimated to be close to positions chr2:106,476,853 and chr1:163,466,814. Portions of the derivative (der) chromosomes are depicted in blue, while reference (ref) chromosomal regions are portrayed in yellow. Fluorescent labels (optical tags) are indicated by vertical lines within the schematized ref and der chromosomal regions, which align with the labeling pattern found in individual molecules of ultra-high molecular weight DNA. The labeling pattern with the transgene indicates that 5 full-length copies integrated in a tail-to-head orientation.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/4f1700222ad2fbbb3059fdfd.png"},{"id":106311003,"identity":"87ef41ca-92af-445e-8dea-dd577cf96298","added_by":"auto","created_at":"2026-04-07 10:27:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75244,"visible":true,"origin":"","legend":"\u003cp\u003eCopy number variant analysis of the optical genome mapping data indicates regions of monosomy exist within chr1 and chr2 in the mutant genome of Line 781. A lower density of labeled molecules in the mutant genome indicates regions in chr1 (3.9 Mbp) and chr2 (1.8 Mbp) where the copy number (CN) is only one (red highlighted regions), instead of the normal value of two (faint gray middle line). Termini of these regions correspond with the breakpoints in the derivative chromosomes indicating that segments of chr1 and chr2 were lost during the RT, thus the translocation is unbalanced.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/70b797101ddb6da91c15558c.png"},{"id":106311075,"identity":"962c35cd-0450-4f82-a04e-0db7b0fd953c","added_by":"auto","created_at":"2026-04-07 10:28:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17673,"visible":true,"origin":"","legend":"\u003cp\u003eSanger sequencing of PCR products that cross a junction ascertained the RT breakpoints in the mutant genome of Line 781. The combination of der1-1C and der1-2D primers resulted in the shortest PCR product for der1 (~600 bp), whereas primer pairs der2-2C with Tg-2 and der2-1D with Tg-1 generated the shortest PCR products for der2 (~1.8 and ~2.4 kb, respectively) with the primers listed in Table 1. Sanger sequencing using primers listed in Table 2 determined the sequence of the amplicons. Shown are portions of the DNA sequence obtained for the top strand that cross a breakpoint in der1 and der2. Chr1-specific sequence is displayed in green, while chr2-specific sequence is portrayed in blue; blue underlined sequence denotes insertion of a small piece of inverted chr2 sequence (see Results for more information). Red letters indicate sequence from the \u003cem\u003ePlp1-lacZ\u003c/em\u003e transgene, while black letters signify nucleotides that likely stem from the repair process of the translocation/transgene insertion. Chromosomal positions that abut a junction are indicated in accordance with GRCm38.p6 as the reference genome.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/4893e1452d061169dfb2f8ac.png"},{"id":106311115,"identity":"c09a3474-caf1-46ae-b63b-036244d54111","added_by":"auto","created_at":"2026-04-07 10:28:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1727756,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9271724/v1/9e65825e-db93-48a6-82da-78875addef53.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detection of a complex chromosomal rearrangement in a novel mouse mutant by optical genome mapping","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGenomic rearrangements constitute large-scale structural alterations in DNA ranging in size from just over 50 bp to megabase pairs as the result of duplications, deletions, insertions, inversions, or translocations (for reviews see: Carvalho and Lupski \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Collins and Talkowski \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ho et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Laufer et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While not all constitutional (germline) structural variants (SVs) lead to aberrant phenotypes or health-related issues, a fair number of them give rise to genomic disorders due to gene disruption/dysregulation, copy number variation (CNV) of a dosage-sensitive gene or non-coding \u003cem\u003ecis\u003c/em\u003e-regulatory element, or generation of a fusion gene whose product has altered activity or function (Harel and Lupski \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Spielmann and Klopocki \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Stankiewicz and Lupski \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Weischenfeldt et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A variety of methods exist for detection of novel SVs, from low resolution cytogenetic techniques such as fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) to high resolution techniques such as long-read sequencing, whose utilities are often subject to the type of SV under investigation (De Coster et al. 2019; Ho et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Moustakli et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Porubsky and Eichler \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the newest techniques to be used for discovery of SVs is optical genome mapping (OGM) (Chan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Garcia-Heras \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mantere et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The technology consists of adding an optical/fluorescent tag to a particular target site throughout the genome and measuring the distance between successive tags in each molecule of ultra-high molecular weight DNA (Dremsek et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Levy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The reads are then \u003cem\u003ede novo\u003c/em\u003e assembled locally and compared to a reference genome. OGM has been used to identify balanced and unbalanced reciprocal translocations (RTs) (Lederbogen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Montjean et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ogiwara et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schnause et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) among other types of SVs, and even aid in localizing a transgene integration site in mouse (Ding et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe current study highlights some of the unique attributes of OGM amid discovery of a complex SV marked by a transgene in a mouse line. The transgene uses the mouse myelin proteolipid protein (\u003cem\u003ePlp1\u003c/em\u003e) promoter to drive expression of a bacterial \u003cem\u003elacZ\u003c/em\u003e expression cassette (Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u003cem\u003elacZ\u003c/em\u003e gene has been used widely as a reporter in transgenic mice due to the innocuous nature of its gene product (β-galactosidase) in mammalian cells (Cui et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Our group has generated over twenty transgenic mouse lines using the same \u003cem\u003elacZ\u003c/em\u003e expression cassette without any untoward consequences except in one, Line 781 (Hamdan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wight et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). While correct spatiotemporal expression of the \u003cem\u003ePlp1-lacZ\u003c/em\u003e transgene remains intact in Line 781 (Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), mice hemizygous for the transgene have small body size and paws and dysmorphic facial features compared with their nontransgenic littermates. The mutant phenotype is 100% penetrant with the transgene. Initially, we surmised that integration of the transgene may have disrupted a critical gene in Line 781. However, FISH analysis presented in this study indicates that the chromosome which harbors the transgene also has a nearby reciprocal translocation (RT), which alternatively could trigger the aberrant phenotype. A RT is said to be balanced if there is no net gain or loss of genetic material (Wilch and Morton \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Typically, balanced RTs do not result in an overt phenotype in the carrier unless the translocation dissociates a critical distal regulatory element from a gene sensitive to variations in copy number or transects the gene body. Instead, phenotypic abnormalities are more likely to arise from unbalanced RTs due to deletion (De Gregori et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) or duplication (Howarth et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) of genetic material at the breakpoint junction. In the current study, we used OGM to discern the type of RT present in mutant animals from Line 781. The data revealed that the RT in this line is unbalanced, resulting in the deletion of more than 30 genes from the affected chromosomes combined. Besides determining the approximate location of deleted sequence in the derivative (der) chromosomes, OGM determined that the transgene integrated within a single site of the genome at or close to the breakpoint junction in der2. Due to asymmetric labeling of the transgene sequence by OGM, we were able to determine the number of full copies that integrated, as well as their orientation. Sanger sequencing of PCR products that traverse the RT breakpoints in der2 established that the transgene copies lay directly between the intersection of chromosomal pieces. Thus, it is likely that the underlying etiology in mutant animals from Line 781 may be due to the loss of one or more dosage-sensitive genes from the derivative chromosome(s).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMice from Line 781 were used in the study. Generation of mice which harbor a \u003cem\u003elacZ\u003c/em\u003e transgene [mPLP(+)Z/FL] driven by the mouse \u003cem\u003ePlp1\u003c/em\u003e promoter has been described, previously (Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). All procedures involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences (UAMS) in compliance with the US Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. Mice were produced by breeding hemizygous transgenic (i.e., mutant) males with C57BL/6 wild-type females. The resulting progeny was genotyped for presence of the transgene by PCR analysis with genomic DNA isolated from tail biopsies according to the methods of Truett et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and the \u003cem\u003elacZ\u003c/em\u003e primer pair described by Stratman et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Because the aberrant phenotype in Line 781 is 100% penetrant with the transgene, mice will be distinguished by phenotype\u0026mdash;mutant or wild-type\u0026mdash;instead of genotype, henceforth.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicro-CT analysis\u003c/h3\u003e\n\u003cp\u003eMale mice at 6 months of age were euthanized by isoflurane overdose followed by cervical dislocation to ensure death. Heads were harvested, skin removed, and subsequently fixed in 10% neutral buffered formalin at 4\u0026deg;C for 24h, washed 3 times (1 min, each) in phosphate buffered saline (pH 7.4), and stored long-term in 70% ethanol. Micro-CT images were acquired using the micro-CT 45 machine from SCANCO Medical AG (Br\u0026uuml;ttisellen, Switzerland). Each sample was placed in a 25.2 mm diameter tube and scanned at 55 kV, 72 \u0026micro;A and 4W using a 0.5mm Al filter. A medium resolution scan (voxel size\u0026thinsp;=\u0026thinsp;24.5 \u0026micro;m) of the entire skull was obtained. Coronal and sagittal images were rendered using the RayTracer (SCANCO) software, which generates a 3D reconstruction of the scanned skull.\u003c/p\u003e\n\u003ch3\u003eLongitudinal growth study\u003c/h3\u003e\n\u003cp\u003eBody weight and length was measured weekly in a cohort of mutant and wild-type mice from Line 781 between 3\u0026ndash;16 weeks of age. Mice were lightly anesthetized with 2% isoflurane in a chamber prior to measurement. Body length indicates the distance (cm) from nose to anus. Comparisons were made between mutant and wild-type mice of the same sex. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) at each age for a specific phenotype (genotype). Results were considered statistically significant if the p-value was less than 0.05 using a standard t-test.\u003c/p\u003e\n\u003ch3\u003eFISH analysis\u003c/h3\u003e\n\u003cp\u003eLymphocytes were isolated from the spleen of a mutant mouse from Line 781 at SeeDNA Biotech Inc. (Windsor, Ontario, Canada). The cells were cultured at 37\u0026deg;C in RMPI 1640 medium supplemented with fetal calf serum, concanavalin A, lipopolysaccharide and mercaptoethanol. Subsequently, cells were harvested and chromosomal slides prepared by conventional methods (hypotonic treatment, fixation, and air dry) employed at SeeDNA. Initially, the mPLP(+)Z/FL transgene probe was labeled with digoxin and detected by rhodamine. The procedure for hybridization and detection was performed according to published methods (Heng and Tsui \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Heng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). FISH signals were observed under fluorescent microscopy. Images were captured with a charged-coupled device (CCD) camera and merged using RS Image software. Additional FISH analysis was performed likewise, except the transgene probe was labeled with biotin and detected by fluorescein isothiocyanate (Heng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), in conjunction with mouse chromosome 1 paint probes (Cambio, Cambridge, UK) labeled with Cy3.\u003c/p\u003e\n\u003ch3\u003eOptical genome mapping (OGM)\u003c/h3\u003e\n\u003cp\u003eOGM sample preparation, mapping, and bioinformatics analysis was conducted by the Molecular Genomics core at Texas A\u0026amp;M University (College Station, TX, USA). Ultra-high molecular weight DNA isolated from liver of a mutant female mouse (Line 781) was labeled with DL-green fluorophores at CTTAAG sites with DLE-1 methyltransferase. The label density averaged 15.017 labels per 100 kb. The average length of molecules (DNA fragments) was 246.092 kbp. The contig N50 was 243.616 kbp.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ede novo\u003c/em\u003e assembly pipeline in the Bionano Solve software (version 1.6.1) was used to build the mutant mouse genome from Line 781, with GRCm38.p6 as the reference genome. The rare variant analysis pipeline in the Bionano software was used to map the translocation breakpoints, associated deletions, and transgene integration site/copy number, upon consideration of the transgene sequence (Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which contains several target sites for labeling. The confidence cutoff was 0.95.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePCR and sequencing validation of breakpoints and transgene integration site\u003c/h2\u003e \u003cp\u003ePCR analysis was performed using primers that tile the chromosomal regions in the vicinity of the breakpoints predicted by OGM. Sequences of the primers, as well as a couple of transgene-specific primers, are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The transgene-specific primers (Tg-1 and Tg-2) were fixed (invariable) in the PCR reactions for amplification of products from der2. Chr2-specific sense primers der2-2A to der2-2F were used separately, in conjunction with Tg-2 as the antisense primer. Alternatively, chr1-specific antisense primers der2-1A to der2-1F were used individually, along with the Tg-1 sense primer. To amplify sequences that traverse the RT junction in der1, a chr1-specific sense primer (der1-1A to der1-1F) was paired with a chr2-specific antisense primer (der1-2A to der1-2F). Genomic DNA isolated from the liver of a female mutant mouse (Line 781) was used as a template in the reactions. PCR reactions (25 \u0026micro;L, total) consisted of 50 ng of genomic DNA, primers each at a final concentration of 0.5 \u0026micro;M final, and Platinum SuperFi Master Mix (Invitrogen, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s specifications. PCR conditions were as follows: 30 sec at 95\u0026deg;C (denaturation), 20 sec at 65\u0026deg;C (annealing), and 60 sec at 72\u0026deg;C (extension) for 35 cycles. The PCR products were resolved by gel electrophoresis on a 0.7% agarose gel. PCR was repeated again for those primer pairs that yielded the smallest product across each of the three junctions; der2 has two junctions since the transgene integrated directly at the intersection of the RT. Serial PCR reactions were performed similarly, except that 1.25 \u0026micro;L of the reaction mixture from the first round of PCR was used as template DNA. Products from the serial round of PCR were resolved on a 0.7% agarose gel, bands excised, and DNA purified using the QIAquick Gel Extraction Kit (Qiagen, Germantown, MD, USA) according to the manufacturer\u0026rsquo;s recommendations. DNA sequences of the PCR products were determined using the primers listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, which were named according to the primer source (chr1, chr2, Tg) followed by the target chromosome (der1, der2). Sanger sequencing was performed at GENEWIZ (Azenta Life Sciences, South Plainfield, NJ, USA) and the DNA Sequencing Core Facility at UAMS.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePCR primers* *Tg-2 serves as an antisense primer for der2-2 sense primers; Tg-1 serves as a sense primer for der2-1 antisense primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositions (GRCm38.p6)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GAGAGGCTTATTGGACATCTAAAATATCG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106476542\u0026ndash;106476570\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-2B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CTTAGATCTTCCTTTTATTCGCTGAGAAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106478379\u0026ndash;106478407\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-2C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GAATAGTTCCTTCTAATTTCTCCCTCTACG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106478843\u0026ndash;106478872\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-2D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GCTATAGCTAGCAAATATTCTCCAATCAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106479200\u0026ndash;106479228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-2E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TCATCACTTCTCTATGTCCTGGTGTTTTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106479256\u0026ndash;106479284\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-2F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TGCCACCTCTGTGTAAATGACTAAATGT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106479603\u0026ndash;106479630\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CCGCTATTTCTCTGTTCTCGCTATTATTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-1A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GGTATATCCTATTTTCTCCTTAAGTGCCTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163466837\u0026ndash;163466808\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CTTGAGTTTAGCCAAACAAACTATGGTTAC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163466310\u0026ndash;163466281\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-1C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GAAAGACGGTGAAATACAGTAAGTAGGAAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163466164\u0026ndash;163466135\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-1D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-ATCAAATCAAATTCAGACAGAGACCTGTAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163465279\u0026ndash;163465250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-1E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GTTAGTTAGACAGTGATTTCCCCAAAGATG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163464271\u0026ndash;163464242\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder2-1F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AAGACGGAGGTCAATAAGATGGATATAAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163463780\u0026ndash;163463752\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CTACCTGTGTCTATGATTCTGCTTTCATTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-1A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GTCTTATATTATCAAAGGACTGCTTCCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159563576\u0026ndash;159563603\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CTGAGAATTATGGGTGGATATTTTGGATTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159565280\u0026ndash;159565309\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-1C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TAATTTTCCTGCACTCACTTACCAAGG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159565724\u0026ndash;159565750\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-1D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-ATAACTAAGGCACAGGACTGATTGATG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159566159\u0026ndash;159566185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-1E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CAATATGGTATCTATGAAAGCCTGTTTCCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159567660\u0026ndash;159567689\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-1F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CACTCTTTCCTCAAAGGTTATTTTCTAGGG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159568646\u0026ndash;159568675\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GGATATTTTCTGAGCACCTTCTTTTTCTCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108257618\u0026ndash;108257589\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-2B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CATTGTTATTGGTGTGTATGCTCTGATG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108257149\u0026ndash;108257122\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-2C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AGATTATTACCAAGAGCACTCCCTGTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108256829\u0026ndash;108256803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-2D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TTGAACTTTTGATTCTACTGTCTCCAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108255484\u0026ndash;108255458\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-2E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CTTACTTTCTATTATTCCCACCACCTCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108255169\u0026ndash;108255142\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eder1-2F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GAGCAAACACCTATTTACCACAGATATTAC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108254391\u0026ndash;108254362\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDNA sequencing primers* *Tg-2 primers are set in the bottom strand of DNA; Tg-1 primers are set in the top strand of DNA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositions (GRCm38.p6)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2A-der2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GCACTGGCATTTTAGAAGG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106479090\u0026ndash;106479108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2B-der2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TCCATGAAACTACTCAAGTGTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:106479125\u0026ndash;106479146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TGTCCAAACTCATCAATGTATCTT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-2B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AGGATTCAAGAACCCCTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-2C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CATAAAGAGAAGATGGAGCCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr1A-der2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-ATTGTGGGGACCTACTTGCTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163465165\u0026ndash;163465145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr1B-der2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AGCCACCACAACATGCAA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:163465074\u0026ndash;163465057\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-1A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CCAGTAAACAGTGTGCTTCATGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AGGATCAGGAGAGTCAGTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTg-1C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GCTGGGAAATGACAGAATGCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr1A-der1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TTTTCCTGCACTCACTTACC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:159565727\u0026ndash;159565746\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2A-der1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TTGATTCTACTGTCTCCAGCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:108255476\u0026ndash;108255456\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTransgenic mice from Line 781 display a mutant phenotype\u003c/h2\u003e \u003cp\u003eWhile generating mice that contain a \u003cem\u003ePlp1-lacZ\u003c/em\u003e transgene one of the founders displayed craniofacial abnormalities and was smaller in size. Despite this, the founder was fertile and mouse Line 781 was established from him (Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Interestingly, the mutant phenotype is 100% penetrant with the transgene. Therefore, we have elected to differentiate the mice based on phenotype (mutant or wild-type) instead of genotype. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that mutant animals at 6 months of age exhibit smaller body length and paws, and craniofacial aberrations including more prominent cranial sutures, smaller orbital fossae, and a compressed snout that is less pointed compared with wild-type mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo quantitate the diminished size of the mutant longitudinally, body weight and length (nose to anus) was measured weekly in a cohort of mutant and wild-type mice from Line 781 between 3\u0026ndash;16 weeks of age. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, both weight and length of the mutants were significantly decreased compared with wild-type (same sex) counterparts, at all ages examined. Thus, the small size of the mutant originates early in development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFISH analysis indicates mutant mice from Line 781 have a RT\u003c/h2\u003e \u003cp\u003eInitially, we suspected that insertion of the transgene must have disrupted a critical gene. Hence, FISH analysis was performed with lymphocytes isolated from the spleen of a mutant mouse to determine the chromosomal location of the transgene. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the transgene integrated in chr2-specific sequence, but this chromosome\u0026mdash;der2\u0026mdash;also has a RT with chr1, close to the transgene integration site. This explains the reduction in litter size (~\u0026thinsp;3 mice/litter; data not shown) by ~\u0026thinsp;50% when a mutant animal (hemizygous for the transgene) is mated with a wild-type mouse, due to unbalanced gametes in the carrier of the RT (Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, the mutant phenotype is likely due to the RT itself.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOGM reveals that the RT in Line 781 is unbalanced\u003c/h2\u003e \u003cp\u003eTo better assess the RT breakpoints and location of the transgene in Line 781, OGM was performed with ultra-high molecular weight genomic DNA obtained from a mutant female mouse, with an average length of 246.092 kbp. The DNA was labeled with DL-green fluorophores at CTTAAG sites with DLE-1 methyltransferase. Because the target site is palindromic, both DNA strands get labeled. The label density averaged 15.017 labels per 100 kb. The labeled DNA was analyzed on the Bionano Saphyr\u0026reg; system to measure the distance between labels (tags) for each single, extremely long strand of genomic DNA (\u0026lsquo;molecule\u0026rsquo;). The resulting data was entered into the \u003cem\u003ede novo\u003c/em\u003e assembly pipeline to group the molecules by label pattern similarity in order to create maps, which then were used to construct the mutant genome. The rare variant analysis pipeline was used to compare the assembly against a reference genome (GRCm38.p6). Areas of discrepancy within the mutant genome were identified using the structural variant (SV) caller. The SV caller indicated that the RT junction in der2 was separated by extraneous DNA. Based on the array of target sites for labeling within the transgene sequence, the extraneous DNA was determined to be the transgene. It is estimated that five complete copies of the transgene integrated in a tail-to-head orientation, directly between the breakpoints in der2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the breakpoints in der2 were estimated to be in the vicinity of positions chr2:106,476,853 and chr1:163,466,814. The SV caller also estimated the RT junction in der1 occurs near positions chr1:159,566,643 and chr2:108,255,381. Taken together, the data suggest that parts of chromosomes 1 and 2 were deleted during the reorganization of the mutant genome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCopy number variant (CNV) analysis was performed for the mutant genome using the Bionano software. CNV analysis quantitates the depth of coverage of a particular region, relative to the expected depth of the entire genome (Sahajpal et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The analysis indicated areas of monosomy within chromosomes 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which correspond to gaps of missing sequence from the derivative chromosomes when the SV caller estimated breakpoints are considered in combination. Thus, the RT is unbalanced, with segments of chr1 and chr2 missing from the derivative chromosomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the RT breakpoints in Line 781\u003c/h2\u003e \u003cp\u003eSanger sequencing of PCR products which traverse junctions between breakpoints (including with the transgene) was used to determine the precise location of chromosomal rearrangements in mutant mice from Line 781. Primers were designed that tile chr1- and chr2-specific regions of the derivative chromosomes near the breakpoints estimated by the SV caller, which reflect the last labeled site that aligns with the reference chromosome prior to the breakpoint. The primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were named according to the derivative chromosome they target (der1 or der2), followed by the chromosome number the sequence was derived from (1 or 2), and a letter (A\u0026ndash;F) to distinguish the individual primers within a given tiled region (\u0026lsquo;A\u0026rsquo; indicates the 5\u0026prime;-most primer on a particular strand). Because the RT breakpoints in der2 are separated by copies of the transgene, transgene-specific primers (Tg-1 and Tg-2) were also generated and used as fixed primers in the PCR reactions for der2. The transgene-specific primers were numbered according to the chromosome targeted and used jointly in PCR reactions to amplify sequences across the junction of full-length copies of the transgene integrated tail-to-head, producing a product of ~\u0026thinsp;456 bp (data not shown). Primer pairs der2-2C in conjunction with Tg-2, and der2-1D in conjunction with Tg-1, generated the shortest PCR products of the tiled primers for der2, while der1-1C together with der1-2D resulted in the smallest size product for der1 (data not shown). Use of the upstream tiled primers resulted in larger size products consistent with their chromosomal location, validating that the amplicons span a breakpoint. The downstream tiled primers did not produce a product, implying that the sequence is absent from the derivative chromosome.\u003c/p\u003e \u003cp\u003ePrimer pairs that generated the smallest PCR product across each of the junctions in the mutant genome were gel purified and sequenced using the primers listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Due to the relatively large size of amplicons generated from der2, multiple primers were needed to fully sequence a strand. The results determined that the transgene integrated between chr2:106,479,179 and chr1:163,464,519 in der2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, partial copies of the transgene are present at both ends of the transgene integration site. Moreover, there is an insertion of 21 bp of inverted chr2 sequence (positions 108,254,948\u0026ndash;108,254,928) located directly between the transgene and chr1 sequence in der2. The junction for der1 is situated between chr1:159,565,980 and chr2:108,255,242 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), directly separated by a 76-bp insertion of inverted chr2 sequence (positions 108,255,235\u0026ndash;108,255,160). Taken together, these results indicate that portions of chr1 (159,565,981\u0026ndash;163,464,518) and chr2 (106,479,180\u0026ndash;108,255,241 except for insertion of inverted 108,255,235\u0026ndash;108,255,160) are missing from the derivative chromosomes in the mutant. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the protein-coding genes that lay within these deleted regions, encompassing 34 genes in chr1 and 4 genes in chr2. Because the breakpoint occurs within intron 1 of \u003cem\u003eTnr\u003c/em\u003e, only the proximal portion of the gene remains in der1. The rest of the genes are completely missing from the derivative chromosomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein-coding genes deleted in der1/der2\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositions (GRCm38.p6)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTnr\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:159523729\u0026ndash;159931729\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTnn\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160085029\u0026ndash;160153692\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMrps14\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160195215\u0026ndash;160201186\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCacybp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160202367\u0026ndash;160212892\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRabgap1l\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160219174\u0026ndash;160793476\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGpr52\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160574717\u0026ndash;160579704\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRc3h1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160906411\u0026ndash;160974976\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSerpinc1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:160978583\u0026ndash;161002543\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eZbtb37\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161002922\u0026ndash;161034864\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGas5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161034601\u0026ndash;161038539\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDars2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161037580\u0026ndash;161070668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCenpl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161070767\u0026ndash;161086724\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlhl20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161088375\u0026ndash;161131508\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnkrd45\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161142447\u0026ndash;161170510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTex50\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161156843\u0026ndash;161159314\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrdx6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161240112\u0026ndash;161251210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTnfsf4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161395438\u0026ndash;161418206\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTnfsf18\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161494657\u0026ndash;161505290\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFasl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161780691\u0026ndash;161788495\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSuco\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161816112\u0026ndash;161876837\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePigc\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161969188\u0026ndash;161973460\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDnm3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:161982450\u0026ndash;162478321\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMettl13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162533671\u0026ndash;162548541\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVamp4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162570515\u0026ndash;162599082\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMyoc\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162639150\u0026ndash;162649694\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrrc2c\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162671785\u0026ndash;162740560\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFmo4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162793188\u0026ndash;162816072\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFmo1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162829561\u0026ndash;162866610\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFmo2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162874317\u0026ndash;162898758\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFmo6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162916551\u0026ndash;162937566\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFmo3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:162953799\u0026ndash;162984528\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMroh9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:163024302\u0026ndash;163085670\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrrx1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:163245119\u0026ndash;163315145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGorab\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr1:163384903\u0026ndash;163403669\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMpped2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr2:106693181\u0026ndash;106868361\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eArl14ep\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr2:106962529\u0026ndash;106974397\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFshb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr2:107055986\u0026ndash;107059651\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKcna4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003echr2:107290589\u0026ndash;107326804\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we report that mutant mice from Line 781 have an unbalanced RT with 5 full-length copies of a \u003cem\u003ePlp1-lacZ\u003c/em\u003e transgene inserted between the translocation junction in der2. Thus far, only a few cases of transgenes integrating at or near a RT breakpoint have been reported (Durkin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Francke et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kasai et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mahon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). This may be an underrepresentation due to investigators opting not to generate transgenic lines from founders with unexpected phenotypes, or to embryonic lethality. In the case of Line 781, the mutant phenotype (small body size and paws, craniofacial abnormalities) does not obfuscate spatiotemporal expression of the transgene (Patyal et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInitially, we surmised that integration of the transgene in Line 781 disrupted a critical gene (or genes), which results in the mutant phenotype. However, FISH analysis established that an RT between chromosomes 1 and 2 exists in the mutant genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which likely elicits the phenotype. Ligation-mediated PCR (Rosenthal and Jones \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) was undertaken to attempt to walk out from the transgene into the flanking chromosomal regions, but only junctions between adjoining copies of transgene were detected (data not shown). Therefore, OGM was employed to narrow down the junctions between different chromosomal regions or with the transgene. While OGM cannot identify chromosomal rearrangements to the nucleotide level, it has much better resolution than FISH (Levy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, CNV analysis of the OGM data was key in establishing that the RT in Line 781 is unbalanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Knowledge gained from the results with OGM was utilized to design primers that tile chromosomal regions surrounding the estimated breakpoints in the mutant genome for use in long-range PCR, at times in conjunction with fixed primers containing transgene sequence. Sanger sequencing of gel-extracted PCR products determined that the actual breakpoints were in fairly close proximity to those predicted by OGM; 663 and 139 bp apart in der1 for chr1 and chr2 segments, respectively, and 2,326 and 2,295 bp apart in der2 for chr2 and chr1 segments, respectively. Sanger sequencing also determined that four other breaks occurred within chr2, which resulted in small portions of inverted chr2 sequence being inserted at the translocation junction and transgene integration site of der1 (76 bp) and der2 (21 bp), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These rearrangements, coupled with insertion of the transgene at the translocation junction of der2, suggest that a series of recombinational events took place during microinjection of the transgene. Perhaps integration of the transgene caused chromothripis of portions of chr1 and chr2 during the repair process. The rearrangements are very stable since the mutant phenotype of the (Line 781) founder has been passed down for many generations. To the best of our knowledge, only one other study (Ding et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) has used OGM to map the location of a transgene in mouse, thus far. It was possible to determine that five full-length copies of the transgene integrated in a tail-to-head orientation in der2 by OGM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) based upon the array of target sites within the transgene sequence, which result in a distinct labeling pattern.\u003c/p\u003e \u003cp\u003eBecause the RT in Line 781 is unbalanced with deletion of approximately 3.9 (chr1) and 1.8 (chr2) Mbp of sequence in the derivative chromosomes, a monosomy exists over these regions in the mutant genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All total; 38 protein-coding genes are located within the deleted sequence (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It is conceivable that the mutant phenotype arises from a haploinsufficiency due to an insufficient amount of gene product from one (or more) of these genes needed to preserve its function (Johnson et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Veitia et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Intriguingly, microdeletion of portions of the chr1 syntenic region have been reported in patients with 1q24 microdeletion syndrome that present with growth deficiency (weight and height), small hands and feet, and dysmorphic facial features (Ashraf et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Burkardt et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lefroy et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nishimura et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Shepherdson et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The condition is inherited in an autosomal dominant fashion (Lefroy et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shepherdson et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), consistent with mutant mice from Line 781. Several reports (Ashraf et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lefroy et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shepherdson et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have ascribed the anomalies in patients with 1q24 deletion syndrome to a monosomy of the noncoding RNA gene, \u003cem\u003eDNM3OS\u003c/em\u003e, which is located on the opposite strand of \u003cem\u003eDMN3\u003c/em\u003e intron 14. However, while \u003cem\u003eDnm3os\u003c/em\u003e knockout mice display skeletal and craniofacial deformities, heterozygous mice with one functional allele were described as phenotypically normal according to the authors (Watanabe et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), although the weight of heterozygotes appeared to trend lower than wild-type mice at 5 months of age (but not at P30) and there appeared to be some shortening of the sagittal axis of the skull in heterozygotes at P60. Thus, deletion of one allele of \u003cem\u003eDnm3os\u003c/em\u003e cannot fully explain the mutant phenotype in Line 781. Interstitial microdeletion of the chr2 syntenic region in humans (11p14.1), in contrast, is associated with ADHD, autism, developmental delay, and obesity (Shinawi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Hence, it is likely that the growth deficiency as well as other aberrations observed in mutant animals from Line 781 are associated with loss of a gene or genes from the affected locus of chr1.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study highlights some of the unique attributes of OGM amid discovery of a complex SV marked by a transgene in mouse Line 781, which causes an aberrant phenotype in mice that harbor the transgene. With OGM we were able to determine the genomic rearrangements present in the mutant genome, which consist of an unbalanced RT between chr1 and chr2 and a corresponding loss of 3.9 (chr1-specific) and 1.8 (chr2-specific) Mbp of sequence from the derivative chromosomes. Furthermore, OGM was able to determine that five full-length copies of transgene integrated directly between the breakpoint junction of der2 in a tail-to-head orientation. In general, OGM can be used to distinguish the type of RT (balanced \u003cem\u003evs\u003c/em\u003e. unbalanced) and the identity of chromosomes involved. In addition, OGM can be used to determine whether a transgene integrated at a single or multiple site(s) in the genome and into which chromosome, as well as the number of full-length copies of transgene that integrated and their orientation provided that the transgene sequence contains an asymmetric distribution of target sites for labeling. Because a variety of enzymes with different targets are available for OGM, the choice of enzyme should center on the transgene sequence under investigation. With respect to the specific genetic rearrangements observed in our mouse mutant, the resulting phenotype is consistent with 1q24 deletion syndrome in humans having an interstitial deletion of the syntenic region from a single autosome of Chr1. Thus, mutant mice from Line 781 may serve as an animal model, in future studies, to explore the molecular and cellular basis of small stature and other anomalies present in patients with 1q24 deletion syndrome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGH and PAW conceptualization. GH data curation, formal analysis, and validation. GH and PAW investigation and visualization. PAW funding acquisition. GH wrote the first draft. PAW reviewed and edited the final revision. All authors discussed the content and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported in part by a Barton Pilot Study award from the College of Medicine at the University of Arkansas for Medical Sciences and an R01 grant (NS106179) from the US National Institute of Neurological Disorders and Stroke. \u003cem\u003eThe content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe OGM dataset is available at Mendeley Data, V2, doi: 10.17632/3h325jhj93.2. https://data.mendeley.com/datasets/3h325jhj93/2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences (UAMS) in compliance with the US Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAshraf T, Collinson MN, Fairhurst J, Wang R, Wilson LC, Foulds N (2015) Two further patients with the 1q24 deletion syndrome expand the phenotype: a possible role for the miR199\u0026ndash;214 cluster in the skeletal features of the condition. 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Comput Struct Biotechnol J 27:2233\u0026ndash;2242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.csbj.2025.05.043\u003c/span\u003e\u003cspan address=\"10.1016/j.csbj.2025.05.043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mammalian-genome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mage","sideBox":"Learn more about [Mammalian Genome](http://link.springer.com/journal/335)","snPcode":"335","submissionUrl":"https://submission.nature.com/new-submission/335/3","title":"Mammalian Genome","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Optical genome mapping, Unbalanced reciprocal translocation, Structural variant, Mouse mutant, FISH analysis, Transgenic mouse","lastPublishedDoi":"10.21203/rs.3.rs-9271724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9271724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHere we highlight the utilities of optical genome mapping (OGM) in determining the genomic rearrangements present in a novel transgenic mouse line (Line 781), which expresses the bacterial \u003cem\u003elacZ\u003c/em\u003e reporter gene under control of the mouse myelin proteolipid protein (\u003cem\u003ePlp1\u003c/em\u003e) promoter. Hemizygous transgenic mice from Line 781 present with a mutant phenotype (documented here) which entails small body size and paws and craniofacial aberrations that are 100% penetrant, whereas their non-transgenic littermates are phenotypically normal. OGM was used to determine that the transgene sits at the intersection of an unbalanced reciprocal translocation between chromosomes 1 and 2, with deletion of approximately 3.9 (chr1) and 1.8 (chr2) Mbp from the rearranged (derivative) chromosomes, thus resulting in a monosomy over these regions in the mutant genome. As well, OGM was able to determine the number of full-length copies of transgene that integrated and their orientation. Sanger sequencing of PCR products that span a junction were used to determine the chromosomal breakpoints and transgene integration site, precisely. The complex chromosomal rearrangements in Line 781 span 38 protein-coding genes that result in the transection of 1 gene from chr1 and deletion of 33 and 4 genes from chr1 and chr2, respectively. The resulting mutant phenotype is consistent with 1q24 deletion syndrome in humans having an interstitial deletion of the syntenic region in Chr1. Thus, our mouse mutant may serve as an animal model, in future studies, to explore the molecular and cellular basis of anomalies present in patients with 1q24 deletion syndrome.\u003c/p\u003e","manuscriptTitle":"Detection of a complex chromosomal rearrangement in a novel mouse mutant by optical genome mapping","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 10:26:13","doi":"10.21203/rs.3.rs-9271724/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-26T17:21:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T21:22:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-21T01:30:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213188536776822273117920703906580545599","date":"2026-04-04T14:36:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15531284394548446833605741130116028327","date":"2026-04-01T23:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-01T13:07:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T07:43:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T06:30:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mammalian Genome","date":"2026-03-30T20:09:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mammalian-genome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mage","sideBox":"Learn more about [Mammalian Genome](http://link.springer.com/journal/335)","snPcode":"335","submissionUrl":"https://submission.nature.com/new-submission/335/3","title":"Mammalian Genome","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eefb2bfa-de99-42af-81e6-f3d4df827586","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-10T07:25:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 10:26:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9271724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9271724","identity":"rs-9271724","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}