Deciphering the role of a SINE-VNTR-Alu retrotransposon polymorphism as a biomarker of Parkinson's disease progression.

SINE-VNTR-Alu (SVA) retrotransposons are transposable elements which represent a source of genetic variation. We previously demonstrated that the presence/absence of a human-specific SVA, termed SVA_67, correlated with the progression of Parkinson's disease (PD). In the present study, we demonstrate that SVA_67 acts as expression quantitative trait loci, thereby exhibiting a strong regulatory effect across the genome using whole genome and transcriptomic data from the Parkinson's progression markers initiative cohort. We further show that SVA_67 is polymorphic for its variable number tandem repeat domain which correlates with both regulatory properties in a luciferase reporter gene assay in vitro and differential expression of multiple genes in vivo. Additionally, this variation's utility as a biomarker is reflected in a correlation with a number of PD progression markers. These experiments highlight the plethora of transcriptomic and phenotypic changes associated with SVA_67 polymorphism which should be considered when investigating the missing heritability of neurodegenerative diseases.

SPAST Intragenic CNVs Lead to Hereditary Spastic Paraplegia via a Haploinsufficiency Mechanism.

The most common form of hereditary spastic paraplegia (HSP), SPG4 is caused by single nucleotide variants and microrearrangements in the SPAST gene. The high percentage of multi-exonic deletions or duplications observed in SPG4 patients is predisposed by the presence of a high frequency of Alu sequences in the gene sequence. In the present study, we analyzed DNA and RNA samples collected from patients with different microrearrangements in SPAST to map gene breakpoints and evaluate the mutation mechanism. The study group consisted of 69 individuals, including 50 SPG4 patients and 19 healthy relatives from 18 families. Affected family members from 17 families carried varying ranges of microrearrangements in the SPAST gene, while one individual had a single nucleotide variant in the 5'UTR of SPAST. To detect the breakpoints of the SPAST gene, long-range PCR followed by sequencing was performed. The breakpoint sequence was detected for five different intragenic SPAST deletions and one duplication, revealing Alu-mediated microhomology at breakpoint junctions resulting from non-allelic homologous recombination in these patients. Furthermore, SPAST gene expression analysis was performed using patient RNA samples extracted from whole blood. Quantitative real-time PCR tests performed in 14 patients suggest no expression of transcripts with microrearrangements in 5 of them. The obtained data indicate that nonsense-mediated decay degradation is not the only mechanism of hereditary spastic paraplegia in patients with SPAST microrearrangements.

Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation.

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

Alu Methylation Patterns in Type 1 Diabetes: A Case-Control Study.

Evidence suggests that genome-wide hypomethylation may promote genomic instability and cellular senescence, leading to chronic complications in people with diabetes mellitus. Limited data are however available on the Alu methylation status in patients with type 1 diabetes (T1D). Methods: We investigated DNA methylation levels and patterns of Alu methylation in the peripheral blood of 36 patients with T1D and 29 healthy controls, matched for age and sex, by using the COmbined Bisulfite Restriction Analysis method (COBRA). Results: Total Alu methylation rate (mC) was similar between patients with T1D and controls (67.3% (64.4-70.9%) vs. 68.0% (62.0-71.1%), p = 0.874). However, patients with T1D had significantly higher levels of the partial Alu methylation pattern (mCuC + uCmC) (41.9% (35.8-45.8%) vs. 36.0% (31.7-40.55%), p = 0.004) compared to healthy controls. In addition, a positive correlation between levels of glycated hemoglobin (HbA1c) and the partially methylated loci (mCuC + uCmC) was observed (Spearman's rho = 0.293, p = 0.018). Furthermore, significant differences were observed between patients with T1D diagnosed before and after the age of 15 years regarding the total methylation mC, the methylated pattern mCmC and the unmethylated pattern uCuC (p = 0.040, p = 0.044 and p = 0.040, respectively). Conclusions: In conclusion, total Alu methylation rates were similar, but the partial Alu methylation pattern (mCuC + uCmC) was significantly higher in patients with T1D compared to healthy controls. Furthermore, this pattern was associated positively with the levels of HbA1c and negatively with the age at diagnosis.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

CircMTA1 promotes glioblastoma angiogenesis by encoding MTA1-134aa.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with rapid angiogenesis. How to inhibit GBM angiogenesis is a key problem to be solved. To explore the targets of inhibiting GBM angiogenesis, this study confirmed that the expression of circMTA1 (hsa_circ_0033614) was significantly upregulated in human brain microvascular endothelial cells exposed to glioma cell-conditioned medium (GECs). The expression of circMTA1 in the cytoplasm was significantly higher than that in the nucleus. Upregulated circMTA1 in GECs can promote cell proliferation, migration, and tube formation. Further exploration of the circularization mechanism of circMTA1 confirmed that KHDRBS1 protein can bind to the upstream and downstream flanking sequences of circMTA1 and promote circMTA1 biogenesis by coordinating Alu element pairing. KHDRBS1 upregulated the proliferation, migration, and tube formation of GECs by promoting the biogenesis of circMTA1. CircMTA1 can encode the protein MTA1-134aa by internal ribosome entry site sequence-mediated translation mechanism, and promote the proliferation, migration, and tube formation of GECs through the encoded MTA1-134aa. This study provides a new target for inhibiting angiogenesis in brain GBM and a new strategy for improving the therapeutic efficacy of GBM.

Alu transposable elements rewire enhancer-promoter network through RNA pairing.

A recent study by Liang et al.1 reveals that interacting enhancer RNAs (eRNAs) and promoter-transcribed upstream antisense RNAs (uaRNAs) can identify enhancer-promoter interactions. Complementary sequences within the interacting eRNAs and uaRNAs, predominantly Alu sequences, confer the specificity for eRNA-uaRNA pairing and hence enhancer-promoter recognition.

Classification of Promoter Sequences from Human Genome.

We have developed a new method for promoter sequence classification based on a genetic algorithm and the MAHDS sequence alignment method. We have created four classes of human promoters, combining 17,310 sequences out of the 29,598 present in the EPD database. We searched the human genome for potential promoter sequences (PPSs) using dynamic programming and position weight matrices representing each of the promoter sequence classes. A total of 3,065,317 potential promoter sequences were found. Only 1,241,206 of them were located in unannotated parts of the human genome. Every other PPS found intersected with either true promoters, transposable elements, or interspersed repeats. We found a strong intersection between PPSs and Alu elements as well as transcript start sites. The number of false positive PPSs is estimated to be 3 × 10-8 per nucleotide, which is several orders of magnitude lower than for any other promoter prediction method. The developed method can be used to search for PPSs in various eukaryotic genomes.

Efficiency of mitochondrial genes and nuclear Alu elements in detecting human DNA in blood meals of Anopheles stephensi mosquitoes: a time-course study.

BACKGROUND: The time required for PCR detection of DNA in human blood meals in vector mosquitoes may vary, depending on the molecular markers used, based on the size and copy number of the amplicons. Detailed knowledge of the blood-feeding behavior of mosquito populations in nature is an essential component for evaluating their vectorial capacity and for assessing the roles of individual vertebrates as potential hosts involved in the transmission of vector-borne diseases. METHODS: Laboratory experiments were conducted to compare the time course of PCR detection of DNA in human blood meals from individual blood-fed Anopheles stephensi mosquitoes, using loci with different characteristics, including two mitochondrial DNA (mtDNA) genes, cytB (228 bp) and 16S ribosomal RNA (rRNA) (157 bp) and nuclear Alu-repeat elements (226 bp) at different time points after the blood meal. RESULTS: Human DNA was detectable up to 84-120 h post-blood-feeding, depending on the length and copy number of the loci. Our results suggest that 16S rRNA and Alu-repeat markers can be successfully recovered from human DNA up to 5 days post-blood-meal. The 16S rDNA and Alu-repeat loci have a significantly (P = 0.008) slower decline rate than the cytB locus. Median detection periods (T50) for the amplicons were 117, 113 and 86.4 h for Alu-repeat, 16S rDNA and cytB, respectively, suggesting an inverse linear relationship between amplicon size/copy number and digestion time. CONCLUSION: This comparative study shows that the Alu-repeat locus is the most efficient marker for time-course identification of human DNA from blood meals in female mosquitoes. It is also a promising tool for determining the anthropophilic index (AI) or human blood index (HBI), i.e. the proportion of blood meals from humans, which is often reported as a relative measure of anthropophagy of different mosquito vectors, and hence a measure of the vector competence of mosquito species collected in the field.

A Genome-Wide Screen for the Exonisation of Reference SINE-VNTR-Alus and Their Expression in CNS Tissues of Individuals with Amyotrophic Lateral Sclerosis.

The hominid-specific retrotransposon SINE-VNTR-Alu (SVA) is a composite element that has contributed to the genetic variation between individuals and influenced genomic structure and function. SVAs are involved in modulating gene expression and splicing patterns, altering mRNA levels and sequences, and have been associated with the development of disease. We evaluated the genome-wide effects of SVAs present in the reference genome on transcript sequence and expression in the CNS of individuals with and without the neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS). This study identified SVAs in the exons of 179 known transcripts, several of which were expressed in a tissue-specific manner, as well as 92 novel exonisation events occurring in the motor cortex. An analysis of 65 reference genome SVAs polymorphic for their presence/absence in the ALS consortium cohort did not identify any elements that were significantly associated with disease status, age at onset, and survival. However, there were transcripts, such as transferrin and HLA-A, that were differentially expressed between those with or without disease, and expression levels were associated with the genotype of proximal SVAs. This study demonstrates the functional consequences of several SVA elements altering mRNA splicing patterns and expression levels in tissues of the CNS.

Complementary Alu sequences mediate enhancer-promoter selectivity.

Enhancers determine spatiotemporal gene expression programs by engaging with long-range promoters1-4. However, it remains unknown how enhancers find their cognate promoters. We recently developed a RNA in situ conformation sequencing technology to identify enhancer-promoter connectivity using pairwise interacting enhancer RNAs and promoter-derived noncoding RNAs5,6. Here we apply this technology to generate high-confidence enhancer-promoter RNA interaction maps in six additional cell lines. Using these maps, we discover that 37.9% of the enhancer-promoter RNA interaction sites are overlapped with Alu sequences. These pairwise interacting Alu and non-Alu RNA sequences tend to be complementary and potentially form duplexes. Knockout of Alu elements compromises enhancer-promoter looping, whereas Alu insertion or CRISPR-dCasRx-mediated Alu tethering to unregulated promoter RNAs can create new loops to homologous enhancers. Mapping 535,404 noncoding risk variants back to the enhancer-promoter RNA interaction maps enabled us to construct variant-to-function maps for interpreting their molecular functions, including 15,318 deletions or insertions in 11,677 Alu elements that affect 6,497 protein-coding genes. We further demonstrate that polymorphic Alu insertion at the PTK2 enhancer can promote tumorigenesis. Our study uncovers a principle for determining enhancer-promoter pairing specificity and provides a framework to link noncoding risk variants to their molecular functions.

Evaluation of DNA Methylation Profiles of LINE-1, Alu and Ribosomal DNA Repeats in Human Cell Lines Exposed to Radiofrequency Radiation.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as "possibly carcinogenic"; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

Disruption of FBN1 by an Alu element insertion: A novel genetic cause of Marfan syndrome.

Alu elements are retrotransposons with ubiquitous presence in the human genome that have contributed to human genomic diversity and health. These approximately 300-bp sequences can cause or mediate disease by disrupting coding/splicing regions in the germline, by insertional mutagenesis in somatic cells, and in promoting formation of copy-number variants. Alu elements may also disrupt epigenetic regulation by affecting non-coding regulatory regions. There are increasing reports of apparently sporadic and inherited genetic disorders caused by Alu-related gene disruption, but Marfan syndrome resulting from Alu element insertion has not been previously described. We report a family with classic features of Marfan syndrome whose previous FBN1 genetic testing was inconclusive. Using contemporary next-generation sequencing and bioinformatics analysis, a pathogenic/disruptive Alu insertion occurring in the coding region of the FBN1 gene was identified (c.6564_6565insAlu; p. Glu2189fs) and was confirmed and specified further with Sanger sequencing. This identified the molecular basis of disease in the family that was missed using previous genetic testing technologies and highlights a novel pathogenic mechanism for Marfan syndrome. This case adds to the growing literature of Mendelian diseases caused by Alu retrotransposition, and it also shows the growing capability of genomic technologies for detecting atypical mutation events.

Alu RNA fold links splicing with signal recognition particle proteins.

Transcriptomic diversity in primates was considerably expanded by exonizations of intronic Alu elements. To better understand their cellular mechanisms we have used structure-based mutagenesis coupled with functional and proteomic assays to study the impact of successive primate mutations and their combinations on inclusion of a sense-oriented AluJ exon in the human F8 gene. We show that the splicing outcome was better predicted by consecutive RNA conformation changes than by computationally derived splicing regulatory motifs. We also demonstrate an involvement of SRP9/14 (signal recognition particle) heterodimer in splicing regulation of Alu-derived exons. Nucleotide substitutions that accumulated during primate evolution relaxed the conserved left-arm AluJ structure including helix H1 and reduced the capacity of SRP9/14 to stabilize the closed Alu conformation. RNA secondary structure-constrained mutations that promoted open Y-shaped conformations of the Alu made the Alu exon inclusion reliant on DHX9. Finally, we identified additional SRP9/14 sensitive Alu exons and predicted their functional roles in the cell. Together, these results provide unique insights into architectural elements required for sense Alu exonization, identify conserved pre-mRNA structures involved in exon selection and point to a possible chaperone activity of SRP9/14 outside the mammalian signal recognition particle.

Long interspersed nuclear element 1 and B1/Alu repeats blueprint genome compartmentalization.

The organization of the genome into euchromatin and heterochromatin has been known for almost 100 years [1]. More than 50% of mammalian genomes contain repetitive sequences [2,3]. Recently, a functional link between the genome and its folding has been identified [4,5]. Homotypic clustering of long interspersed nuclear element 1 (LINE1 or L1) and B1/Alu retrotransposons forms grossly exclusive nuclear domains that characterize and predict heterochromatin and euchromatin, respectively. The spatial segregation of L1 and B1/Alu-rich compartments is conserved in mammalian cells and can be rebuilt during the cell cycle and established de novo in early embryogenesis. Inhibition of L1 RNA drastically weakened homotypic repeat contacts and compartmental segregation, indicating that L1 plays a more significant role than just being a compartmental marker. This simple and inclusive genetic coding model of L1 and B1/Alu in shaping the macroscopic structure of the genome provides a plausible explanation for the remarkable conservation and robustness of its folding in mammalian cells. It also proposes a conserved core structure on which subsequent dynamic regulation takes place.

Epigenetic Gene-Regulatory Loci in Alu Elements Associated with Autism Susceptibility in the Prefrontal Cortex of ASD.

Alu elements are transposable elements that can influence gene regulation through several mechanisms; nevertheless, it remains unclear whether dysregulation of Alu elements contributes to the neuropathology of autism spectrum disorder (ASD). In this study, we characterized transposable element expression profiles and their sequence characteristics in the prefrontal cortex tissues of ASD and unaffected individuals using RNA-sequencing data. Our results showed that most of the differentially expressed transposable elements belong to the Alu family, with 659 loci of Alu elements corresponding to 456 differentially expressed genes in the prefrontal cortex of ASD individuals. We predicted cis- and trans-regulation of Alu elements to host/distant genes by conducting correlation analyses. The expression level of Alu elements correlated significantly with 133 host genes (cis-regulation, adjusted p < 0.05) associated with ASD as well as the cell survival and cell death of neuronal cells. Transcription factor binding sites in the promoter regions of differentially expressed Alu elements are conserved and associated with autism candidate genes, including RORA. COBRA analyses of postmortem brain tissues showed significant hypomethylation in global methylation analyses of Alu elements in ASD subphenotypes as well as DNA methylation of Alu elements located near the RNF-135 gene (p < 0.05). In addition, we found that neuronal cell density, which was significantly increased (p = 0.042), correlated with the expression of genes associated with Alu elements in the prefrontal cortex of ASD. Finally, we determined a relationship between these findings and the ASD severity (i.e., ADI-R scores) of individuals with ASD. Our findings provide a better understanding of the impact of Alu elements on gene regulation and molecular neuropathology in the brain tissues of ASD individuals, which deserves further investigation.

Framework of the Alu Subfamily Evolution in the Platyrrhine Three-Family Clade of Cebidae, Callithrichidae, and Aotidae.

The history of Alu retroposons has been choreographed by the systematic accumulation of inherited diagnostic nucleotide substitutions to form discrete subfamilies, each having a distinct nucleotide consensus sequence. The oldest subfamily, AluJ, gave rise to AluS after the split between Strepsirrhini and what would become Catarrhini and Platyrrhini. The AluS lineage gave rise to AluY in catarrhines and to AluTa in platyrrhines. Platyrrhine Alu subfamilies Ta7, Ta10, and Ta15 were assigned names based on a standardized nomenclature. However, with the subsequent intensification of whole genome sequencing (WGS), large scale analyses to characterize Alu subfamilies using the program COSEG identified entire lineages of subfamilies simultaneously. The first platyrrhine genome with WGS, the common marmoset (Callithrix jacchus; [caljac3]), resulted in Alu subfamily names sf0 to sf94 in an arbitrary order. Although easily resolved by alignment of the consensus sequences, this naming convention can become increasingly confusing as more genomes are independently analyzed. In this study, we reported Alu subfamily characterization for the platyrrhine three-family clade of Cebidae, Callithrichidae, and Aotidae. We investigated one species/genome from each recognized family of Callithrichidae and Aotidae and of both subfamilies (Cebinae and Saimiriinae) of the family Cebidae. Furthermore, we constructed a comprehensive network of Alu subfamily evolution within the three-family clade of platyrrhines to provide a working framework for future research. Alu expansion in the three-family clade has been dominated by AluTa15 and its derivatives.

Genomic diversity and differentiation of Alu insertion polymorphisms in a native British and four South Asian migrant populations.

BACKGROUND: Alu insertions are bi-allelic and primate-specific, this makes them a useful marker for studying genetic variation, migration patterns, forensic analyses, paternity, and evolutionary heritage; however, specific population studies are limited. AIM: The objective of this study is to document the level and extent of genetic variation at 39 different Alu loci in five populations (British, Indian Punjabi, Indian Gujarati, Pakistani, and Bangladeshi) from the East Midlands region of the UK. Genetic data on migrant populations is currently limited. SUBJECTS AND METHODS: DNA samples (n = 543) were analysed for 39 Alu insertion polymorphisms using specific primers and standard protocols. Data were analysed for population and forensic genetic parameters. RESULTS: All studied Alus were polymorphic in the British White population while South Asian migrant populations had a variable number of loci which were monomorphic. Highest heterozygosities and lowest match probabilities were observed in the British sample, while the Bangladeshi sample had the lowest heterozygosity and higher match probability. CONCLUSION: The analysed Alus insertions (TPA25, Ya5NBC123, Ya5NBC182, Ya5NBC241, and Ya5NBC242) are highly polymorphic and variable among migrant populations. These loci could be useful for population genomic and differentiation studies.