Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.

Nanopore Sequencing Enables Comprehensive Transposable Element Epigenomic Profiling.

Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.

ZNF91 is an endogenous repressor of the molecular phenotype associated with X-linked dystonia-parkinsonism (XDP).

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder resulting from an inherited intronic SINE-Alu-VNTR (SVA) retrotransposon in the TAF1 gene that causes dysregulation of TAF1 transcription. The specific mechanism underlying this dysregulation remains unclear, but it is hypothesized to involve the formation of G-quadruplexes (G4) structures within the XDP-SVA that impede transcription. In this study, we show that ZNF91, a critical repressor of SVA retrotransposons, specifically binds to G4-forming DNA sequences. Further, we found that genetic deletion of ZNF91 exacerbates the molecular phenotype associated with the XDP-SVA insertion in patient cells, while no difference was observed when ZNF91 was deleted from isogenic control cells. Additionally, we observed a significant age-related reduction in ZNF91 expression in whole blood and brain, indicating a progressive loss of repression of the XDP-SVA in XDP. These findings indicate that ZNF91 plays a crucial role in controlling the molecular phenotype associated with XDP. Since ZNF91 binds to G4-forming DNA sequences in SVAs, this suggests that interactions between ZNF91 and G4-forming sequences in the XDP-SVA minimize the severity of the molecular phenotype. Our results showing that ZNF91 expression levels significantly decrease with age provide a potential explanation for the age-related progressive neurodegenerative character of XDP. Collectively, our study provides important insights into the protective role of ZNF91 in XDP pathogenesis and suggests that restoring ZNF91 expression, destabilization of G4s, or targeted repression of the XDP-SVA could be future therapeutic strategies to prevent or treat XDP.

Adjuvant screening of the Senecavirus A inactivated vaccine in mice and evaluation of its immunogenicity in pigs.

BACKGROUND: Senecavirus A (SVA) causes an emerging vesicular disease (VD) with clinical symptoms indistinguishable from other vesicular diseases, including vesicular stomatitis (VS), foot-and-mouth disease (FMD), and swine vesicular disease (SVD). Currently, SVA outbreaks have been reported in Canada, the U.S.A, Brazil, Thailand, Vietnam, Colombia, and China. Based on the experience of prevention and control of FMDV, vaccines are the best means to prevent SVA transmission. RESULTS: After preparing an SVA inactivated vaccine (CH-GX-01-2019), we evaluated the immunogenicity of the SVA inactivated vaccine mixed with Imject® Alum (SVA + AL) or Montanide ISA 201 (SVA + 201) adjuvant in mice, as well as the immunogenicity of the SVA inactivated vaccine combined with Montanide ISA 201 adjuvant in post-weaned pigs. The results of the mouse experiment showed that the immune effects in the SVA + 201 group were superior to that in the SVA + AL group. Results from pigs immunized with SVA inactivated vaccine combined with Montanide ISA 201 showed that the immune effects were largely consistent between the SVA-H group (200 µg) and SVA-L group (50 µg); the viral load in tissues and blood was significantly reduced and no clinical symptoms occurred in the vaccinated pigs. CONCLUSIONS: Montanide ISA 201 is a better adjuvant choice than the Imject® Alum adjuvant in the SVA inactivated vaccine preparation, and the CH-GX-01-2019 SVA inactivated vaccine can provide effective protection for pigs.

ZBP1 inhibits the replication of Senecavirus A by enhancing NF-κB signaling pathway mediated antiviral response in porcine alveolar macrophage 3D4/21 cells.

BACKGROUND: Senecavirus A (SVA) caused porcine idiopathic vesicular disease (PIVD) showing worldwide spread with economic losses in swine industry. Although some progress has been made on host factors regulating the replication of SVA, the role of Z-DNA binding protein 1 (ZBP1) remains unclear. METHODS: The expression of ZBP1 in SVA-infected 3D/421 cells was analyzed by quantitative real-time PCR (qRT-PCR) and western blot. Western blot and qRT-PCR were used to detect the effects of over and interference expression of ZBP1 on SVA VP2 gene and protein. Viral growth curves were prepared to measure the viral proliferation. The effect on type I interferons (IFNs), interferon-stimulated genes (ISGs), and pro-inflammatory cytokines in SVA infection was analyzed by qRT-PCR. Western blot was used to analysis the effect of ZBP1 on NF-κB signaling pathway and inhibitor are used to confirm. RESULTS: ZBP1 is shown to inhibit the replication of SVA by enhancing NF-κB signaling pathway mediated antiviral response. SVA infection significantly up-regulated the expression of ZBP1 in 3D4/21 cells. Infection of cells with overexpression of ZBP1 showed that the replication of SVA was inhibited with the enhanced expression of IFNs (IFN-α, IFN-ß), ISGs (ISG15, PKR, and IFIT1) and pro-inflammatory cytokines (IL-6, IL-8, and TNF-α), while, infected-cells with interference expression of ZBP1 showed opposite effects. Further results showed that antiviral effect of ZBP1 is achieved by activation the NF-κB signaling pathway and specific inhibitor of NF-κB also confirmed this. CONCLUSIONS: ZBP1 is an important host antiviral factor in SVA infection and indicates that ZBP1 may be a novel target against SVA.

Right Sinus of Valsalva Aneurysm Rupturing into the Right Atrium and Dissecting into the Interventricular Septum.

The right sinus of the Valsalva aneurysm (SVA) rupturing into the right atrium (RA) and dissecting into the interventricular septum (IVS) is rare. The disease can be definitively diagnosed using two-dimensional (2D) echocardiography and color Doppler ultrasonography. Real-time biplane imaging and three-dimensional (3D) echocardiography offer new perspectives for viewing and diagnosing this disease.

Retroelement-derived RNA and its role in the brain.

Comprising ~40% of the human genome, retroelements are mobile genetic elements which are transcribed into RNA, then reverse-transcribed into DNA and inserted into a new site in the genome. Retroelements are referred to as "genetic parasites", residing among host genes and relying on host machinery for transcription and evolutionary propagation. The healthy brain has the highest expression of retroelement-derived sequences compared to other somatic tissue, which leads to the question: how does retroelement-derived RNA influence human traits and cellular states? While the functional importance of upregulating retroelement expression in the brain is an active area of research, RNA species derived from retroelements influence both self- and host gene expression by contributing to chromatin remodeling, alternative splicing, somatic mosaicism and translational repression. Here, we review the emerging evidence that the functional importance of RNA derived from retroelements is multifaceted. Retroelements can influence organismal states through the seeding of epigenetic states in chromatin, the production of structured RNA and even catalytically active ribozymes, the generation of cytoplasmic ssDNA and RNA/DNA hybrids, the production of viral-like proteins, and the generation of somatic mutations. Comparative sequencing suggests that retroelements can contribute to intraspecies variation through these mechanisms to alter transcript identity and abundance. In humans, an increasing number of neurodevelopmental and neurodegenerative conditions are associated with dysregulated retroelements, including Aicardi-Goutieres syndrome (AGS), Rett syndrome (RTT), Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), multiple sclerosis (MS), schizophrenia (SZ), and aging. Taken together, these concepts suggest a larger functional role for RNA derived from retroelements. This review aims to define retroelement-derived RNA, discuss how it impacts the mammalian genome, as well as summarize data supporting phenotypic consequences of this unique RNA subset in the brain.

SVA Retrotransposons and a Low Copy Repeat in Humans and Great Apes: A Mobile Connection.

Segmental duplications (SDs) constitute a considerable fraction of primate genomes. They contribute to genetic variation and provide raw material for evolution. Groups of SDs are characterized by the presence of shared core duplicons. One of these core duplicons, low copy repeat (lcr)16a, has been shown to be particularly active in the propagation of interspersed SDs in primates. The underlying mechanisms are, however, only partially understood. Alu short interspersed elements (SINEs) are frequently found at breakpoints and have been implicated in the expansion of SDs. Detailed analysis of lcr16a-containing SDs shows that the hominid-specific SVA (SINE-R-VNTR-Alu) retrotransposon is an integral component of the core duplicon in Asian and African great apes. In orang-utan, it provides breakpoints and contributes to both interchromosomal and intrachromosomal lcr16a mobility by inter-element recombination. Furthermore, the data suggest that in hominines (human, chimpanzee, gorilla) SVA recombination-mediated integration of a circular intermediate is the founding event of a lineage-specific lcr16a expansion. One of the hominine lcr16a copies displays large flanking direct repeats, a structural feature shared by other SDs in the human genome. Taken together, the results obtained extend the range of SVAs' contribution to genome evolution from RNA-mediated transduction to DNA-based recombination. In addition, they provide further support for a role of circular intermediates in SD mobilization.

Infectious Recombinant Senecavirus A Expressing p16INK4A Protein.

Senecavirus A (SVA) is an oncolytic RNA virus, and it is the ideal oncolytic virus that can be genetically engineered for editing. However, there has not been much exploration into creating SVA viruses that carry antitumor genes to increase their oncolytic potential. The construction of SVA viruses carrying antitumor genes that enhance oncolytic potential has not been fully explored. In this study, a recombinant SVA-CH-01-2015 virus (p15A-SVA-clone) expressing the human p16INK4A protein, also known as cell cycle-dependent protein kinase inhibitor 2A (CDKN2A), was successfully rescued and characterized. The recombinant virus, called SVA-p16, exhibited similar viral replication kinetics to the parent virus, was genetically stable, and demonstrated enhanced antitumor effects in Ishikawa cells. Additionally, another recombinant SVA virus carrying a reporter gene (iLOV), SVA-iLOV, was constructed and identified using the same construction method as an auxiliary validation. Collectively, this study successfully created a new recombinant virus, SVA-p16, that showed increased antitumor effects and could serve as a model for further exploring the antitumor potential of SVA as an oncolytic virus.

Repeat-Associated Non-AUG Translation of AGAGGG Repeats that Cause X-Linked Dystonia-Parkinsonism.

BACKGROUND: X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE-VNTR-Alu (SVA) retrotransposon carrying an (AGAGGG)n repeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive. OBJECTIVES: We investigated whether (AGAGGG)n repeats undergo repeat-associated non-AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases. METHODS: XDP-specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS-7 cells to determine the subcellular localization of one DPR. RESULTS: We detected putative DPRs from two reading frames, supporting the translation of poly-(Glu-Gly) and poly-(Arg-Glu) species. XDP RAN translation initiates within the (AGAGGG)n sequence and poly-(Glu-Gly) DPRs formed nuclear inclusions in transfected cells. CONCLUSIONS: In summary, our work provides the first in-vitro proof of principle that the XDP-linked (AGAGGG)n repeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Relationship between TIA minus C0-7 angle and C2-7 SVA: analysis of 113 symptomatic patients.

BACKGROUND: Measurement of T1 slope (T1S) can be difficult due to the anatomical positioning of the shoulders. And thoracic inlet angle (TIA) was a morphological parameter and not changed by the position. We proposed a new parameter, TIA minus C0-7 angle (TIA-C07), to evaluate C2-7 SVA in order to overcome the T1S imperfection. METHODS: This was a retrospective radiological analysis of symptomatic subjects. The following cervical parameters were measured: Cervical lordosis angle (CL), C0-7 angle (C0-7), occiput-C2 lordosis angle (O-C2), C2-7 sagittal vertical axis (C2-7 SVA), TIA and TIA-C07. The Pearson correlation test was calculated, and the stepwise multiple regression analysis was conducted to determine the best predictor for C2-7 SVA. A paired sample t test was used to compare the predicted and measured C2-7 SVA. RESULTS: The mean age of 113 patients was 60.02 ± 9.67. The average O-C2, CL, C0-7, TIA, TIA-C07 and C2-C7 SVA was 29.24 ± 8.48°, 13.67 ± 11.22°, 42.91 ± 11.44°, 76.07 ± 9.54°, 33.16 ± 13.18° and 21.34 ± 11.42 mm. The predictive formula was founded: C2-7 SVA = 2.80 + 0.56 * (TIA-C07) (R = 0.645, R2 = 0.416). There was no statistical difference between the predicted and the measured C2-7 SVA (t = 0.085, P = 0.933). CONCLUSIONS: TIA and C0-7 mismatch may significantly impact cervical alignment, and a greater T1A-C07 was related to a greater degree of C2-7 SVA. TIA-C07 may be a more important predictor for C2-7 SVA.

Association between sagittal spinal alignment and postoperative shoulder range of motion following reverse total shoulder arthroplasty.

BACKGROUND: The range of internal rotation sometimes deteriorates after reverse total shoulder arthroplasty; however, the underlying mechanisms remain unclear. This study aimed to investigate the association between sagittal spinal alignment and internal rotation deterioration after reverse total shoulder arthroplasty. METHODS: We retrospectively reviewed 52 patients who underwent reverse total shoulder arthroplasty by a single surgeon between October 2014 and December 2018. The sagittal spine was radiographed, and the sagittal spinal alignment was evaluated based on 10 parameters (e.g. C7-HA: the distance between the plumb line from the center of the seventh cervical vertebral body and the center of the hip axis). We divided the patients into two groups, group A (internal rotation deterioration; 23 patients) and B (without internal rotation deterioration; 29 patients). Logistic regression analysis was performed to analyze the association between sagittal spinal alignment and internal rotation deterioration after reverse total shoulder arthroplasty, and a receiver operating characteristic curve was used to analyse the cutoff value of independent variables that was correlated with IR deterioration. RESULTS: Logistic regression analysis demonstrated the correlation between C7-HA and internal rotation deterioration (odds ratio, 1.95; 95% confidence interval, 1.33-2.84; P < 0.001). The receiver operating characteristic curve suggested that when C7-HA was at least 2.44 cm, it was strongly correlated with deterioration of internal rotation (sensitivity, 91.3%; specificity, 93.1%; P < 0.001; area under the curve, 0.938). Group A was significantly inferior to group B in not only internal rotation but also flexion and abduction. CONCLUSIONS: This study suggested that a remarkably positive value in C7-HA was a factor that was correlated with deterioration of internal rotation. In addition, the deformation of sagittal spinal alignment would impact on the range of motion after reverse total shoulder arthroplasty.

Transcriptional Alterations in X-Linked Dystonia-Parkinsonism Caused by the SVA Retrotransposon.

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.

Insertion of an SVA element in MSH2 as a novel cause of Lynch syndrome.

Germline mutations in the DNA mismatch repair (MMR) genes cause Lynch syndrome (LS). In this study, we identified and characterized a novel SINE-VNTR-Alu (SVA) insertion in exon 12 of MSH2 in an individual with early-onset colorectal cancer and a very strong LS family history. RT-PCR analysis indicated a larger aberrant MSH2 transcript in one of the family members. MSK-IMPACT next-generation sequencing and long-range PCR analyses revealed an insertion in MSH2 exon 12 at the c.1972 position in an antisense orientation. The insertion was further characterized as an SVA element approximately 3 kb in length, belonging to the SVA_F1 family of retrotransposons. This variant also segregated with LS related cancers in four affected family members in this family. Based on this evidence, this MSH2 SVA insertion is considered pathogenic.

A virulent and pathogenic infectious clone of Senecavirus A.

Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinitis pigmentosa, obesity, polydactyly, cognitive impairment and renal failure. Pathogenic variants in 24 genes account for the molecular basis of >80% of cases. Toward saturated discovery of the mutational basis of the disorder, we carefully explored our cohorts and identified a hominid-specific SINE-R/VNTR/Alu type F (SVA-F) insertion in exon 13 of BBS1 in eight families. In six families, the repeat insertion was found in trans with c.1169 T > G, p.Met390Arg and in two families the insertion was found in addition to other recessive BBS loci. Whole genome sequencing, de novo assembly and SNP array analysis were performed to characterize the genomic event. This insertion is extremely rare in the general population (found in 8 alleles of 8 BBS cases but not in >10 800 control individuals from gnomAD-SV) and due to a founder effect. Its 2435 bp sequence contains hallmarks of LINE1 mediated retrotransposition. Functional studies with patient-derived cell lines confirmed that the BBS1 SVA-F is deleterious as evidenced by a significant depletion of both mRNA and protein levels. Such findings highlight the importance of dedicated bioinformatics pipelines to identify all types of variation.

Recent advances on protein-based quantification of extracellular vesicles.

Extracellular vesicles (EVs) are secreted by almost all cells into the circulatory system and have the important function of intercellular communication. Ranging in size from 50 to 1000 nm, they are further classified based on origin, size, physical properties and function. EVs have shown the potential for studying various physiological and pathological processes, such as characterizing their parent cells with molecular markers that could further signify diseases. Proteins within EVs are the building blocks for the vesicles to function within a biological system. Isolation and proteomic profiling of EVs can advance the understanding of their biogenesis and functions, which can give further insight of how they can be used in clinical settings. However, the nanoscale size of EVs, which is much smaller than that of cells, comprises a major challenge for EV isolation and the characterization of their protein cargos. With the recent advances of bioanalytical techniques such as lab-on-a-chip devices and innovated flow cytometry, the quantification of EV proteins from a small number of vesicles down to the single vesicle level has been achieved, shining light on the promising applications of these small vesicles for early disease diagnosis and treatment monitoring. In this article, we first briefly review conventional EV protein determination technologies and their limitations, followed by detailed description and analysis of emerging technologies used for EV protein quantification, including optical, non-optical, microfluidic, and single vesicle detection methods. The pros and cons of these technologies are compared and the current challenges are outlined. Future perspectives and potential research directions of the EV protein analysis methods are discussed.

Sagittal balance of the cervical spine: a systematic review and meta-analysis.

PURPOSE: The purpose of this systematic review and meta-analysis was to compare the cervical sagittal parameters between patients with cervical spine disorder and asymptomatic controls. METHODS: Two independent authors systematically searched online databases including Pubmed, Scopus, Cochrane library, and Web of Science up to June 2020. Cervical sagittal balance parameters, such as T1 slope, cervical SVA (cSVA), and spine cranial angle (SCA), were compared between the cervical spine in healthy, symptomatic, and pre-operative participants. Where possible, we pooled data using random-effects meta-analysis, by CMA software. Heterogeneity and publication bias were assessed using the I-squared statistic and funnel plots, respectively. RESULTS: A total of 102 studies, comprising 13,802 cases (52.7% female), were included in this meta-analysis. We used the Newcastle-Ottawa Scale (NOS) to evaluate the quality of studies included in this review. Funnel plot and Begg's test did not indicate obvious publication bias. The pooled analysis reveals that the mean (SD) values were: T1 slope (degree), 24.5 (0.98), 25.7 (0.99), 25.4 (0.34); cSVA (mm), 18.7 (1.76), 22.7 (0.66), 22.4 (0.68) for healthy population, symptomatic, and pre-operative assessment, respectively. The mean value of the SCA (degree) was 79.5 (3.55) and 75.6 (10.3) for healthy and symptomatic groups, respectively. Statistical differences were observed between the groups (all P values < 0.001). CONCLUSION: The findings showed that the T1 slope and the cSVA were significantly lower among patients with cervical spine disorder compared to controls and higher for the SCA. Further well-conducted studies are needed to complement our findings.

CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region.

SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.

Complex Transposon Insertion as a Novel Cause of Pompe Disease.

Pompe disease (OMIM#232300) is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene. According to public mutation databases, more than 679 pathogenic variants have been described in GAA, none of which are associated with mobile genetic elements. In this article, we report a novel molecular genetic cause of Pompe disease, which could be hardly detected using routine molecular genetic analysis. Whole genome sequencing followed by comprehensive functional analysis allowed us to discover and characterize a complex mobile genetic element insertion deep in the intron 15 of the GAA gene in a patient with infantile onset Pompe disease.