Regulatory SVA retrotransposons and classical HLA genotyped-transcripts associated with Parkinson's disease.

Introduction: Parkinson's disease (PD) is a neurodegenerative and polygenic disorder characterised by the progressive loss of neural dopamine and onset of movement disorders. We previously described eight SINE-VNTR-Alu (SVA) retrotransposon-insertion-polymorphisms (RIPs) located and expressed within the Human Leucocyte Antigen (HLA) genomic region of chromosome 6 that modulate the differential co-expression of 71 different genes including the HLA classical class I and class II genes in a Parkinson's Progression Markers Initiative (PPMI) cohort. Aims and methods: In the present study, we (1) reanalysed the PPMI genomic and transcriptomic sequencing data obtained from whole blood of 1521 individuals (867 cases and 654 controls) to infer the genotypes of the transcripts expressed by eight classical HLA class I and class II genes as well as DRA and the DRB3/4/5 haplotypes, and (2) examined the statistical differences between three different PD subgroups (cases) and healthy controls (HC) for the HLA and SVA transcribed genotypes and inferred haplotypes. Results: Significant differences for 57 expressed HLA alleles (21 HLA class I and 36 HLA class II alleles) up to the three-field resolution and four of eight expressed SVA were detected at p<0.05 by the Fisher's exact test within one or other of three different PD subgroups (750 individuals with PD, 57 prodromes, 60 individuals who had scans without evidence of dopamine deficits [SWEDD]), when compared against a group of 654 HCs within the PPMI cohort and when not corrected by the Bonferroni test for multiple comparisons. Fourteen of 20 significant alleles were unique to the PD-HC comparison, whereas 31 of the 57 alleles overlapped between two or more different subgroup comparisons. Only the expressed HLA-DRA*01:01:01 and -DQA1*03:01:01 protective alleles (PD v HC), the -DQA1*03:03:01 risk (HC v Prodrome) or protective allele (PD v Prodrome), the -DRA*01:01:02 and -DRB4*01:03:02 risk alleles (SWEDD v HC), and the NR_SVA_381 present genotype (PD v HC) at a 5% homozygous insertion frequency near HLA-DPA1, were significant (Pc<0.1) after Bonferroni corrections. The homologous NR_SVA_381 insertion significantly decreased the transcription levels of HLA-DPA1 and HLA-DPB1 in the PPMI cohort and its presence as a homozygous genotype is a risk factor (Pc=0.012) for PD. The most frequent NR_SVA_381 insertion haplotype in the PPMI cohort was NR_SVA_381/DPA1*02/DPB1*01 (3.7%). Although HLA C*07/B*07/DRB5*01/DRB1*15/DQB1*06 was the most frequent HLA 5-loci phased-haplotype (n, 76) in the PPMI cohort, the NR_SVA_381 insertion was present in only six of them (8%). Conclusions: These data suggest that expressed SVA and HLA gene alleles in circulating white blood cells are coordinated differentially in the regulation of immune responses and the long-term onset and progression of PD, the mechanisms of which have yet to be elucidated.

Exploring SVA Insertion Polymorphisms in Shaping Differential Gene Expressions in the Central Nervous System.

Transposable elements (TEs) are repetitive elements which make up around 45% of the human genome. A class of TEs, known as SINE-VNTR-Alu (SVA), demonstrate the capacity to mobilise throughout the genome, resulting in SVA polymorphisms for their presence or absence within the population. Although studies have previously highlighted the involvement of TEs within neurodegenerative diseases, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS), the exact mechanism has yet to be identified. In this study, we used whole-genome sequencing and RNA sequencing data of ALS patients and healthy controls from the New York Genome Centre ALS Consortium to elucidate the influence of reference SVA elements on gene expressions genome-wide within central nervous system (CNS) tissues. To investigate this, we applied a matrix expression quantitative trait loci analysis and demonstrate that reference SVA insertion polymorphisms can significantly modulate the expression of numerous genes, preferentially in the trans position and in a tissue-specific manner. We also highlight that SVAs significantly regulate mitochondrial genes as well as genes within the HLA and MAPT loci, previously associated within neurodegenerative diseases. In conclusion, this study continues to bring to light the effects of polymorphic SVAs on gene regulation and further highlights the importance of TEs within disease pathology.

CRISPR deletion of a SINE-VNTR-Alu (SVA_67) retrotransposon demonstrates its ability to differentially modulate gene expression at the MAPT locus.

Background: SINE-VNTR-Alu (SVA) retrotransposons are hominid-specific elements which have been shown to play important roles in processes such as chromatin structure remodelling and regulation of gene expression demonstrating that these repetitive elements exert regulatory functions. We have previously shown that the presence or absence of a specific SVA element, termed SVA_67, was associated with differential expression of several genes at the MAPT locus, a locus associated with Parkinson's Disease (PD) and frontotemporal dementia. However, we were not able to demonstrate that causation of differential gene expression was directed by the SVA due to lack of functional validation. Methods: We performed CRISPR to delete SVA_67 in the HEK293 cell line. Quantification of target gene expression was performed using qPCR to assess the effects on expression in response to the deletion of SVA_67. Differences between CRISPR edit and control cell lines were analysed using two-tailed t-test with a minimum 95% confidence interval to determine statistical significance. Results: In this study, we provide data highlighting the SVA-specific effect on differential gene expression. We demonstrate that the hemizygous deletion of the endogenous SVA_67 in CRISPR edited cell lines was associated with differential expression of several genes at the MAPT locus associated with neurodegenerative diseases including KANSL1, MAPT and LRRC37A. Discussion: This data is consistent with our previous bioinformatic work of differential gene expression analysis using transcriptomic data from the Parkinson's Progression Markers Initiative (PPMI) cohort. As SVAs have regulatory influences on gene expression, and insertion polymorphisms contribute to interpersonal differences in expression patterns, these results highlight the potential contribution of these elements to complex diseases with potentially many genetic components, such as PD.

SINE-VNTR-Alu retrotransposon insertion as a novel mutational event underlying Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive platelet aggregation disorder caused by mutations in ITGA2B or ITGB3. OBJECTIVES: We aimed to assess the phenotype and investigate the genetic etiology of a GT pedigree. METHODS: A patient with bleeding manifestations and mild mental retardation was enrolled. Complete blood count, coagulation, and platelet aggregation tests were performed. Causal mutations were identified via whole exome and genome sequencing and subsequently confirmed through polymerase chain reaction and Sanger sequencing. The transcription of ITGB3 was characterized using RNA sequencing and reverse transcription polymerase chain reaction. The αⅡb and ß3 biosynthesis was investigated via whole blood flow cytometry and in vitro studies. RESULTS: GT was diagnosed in a patient with defective platelet aggregation. Novel compound heterozygous ITGB3 variants were identified, with a maternal nonsense mutation (c.2222G>A, p.Trp741∗) and a paternal SINE-VNTR-Alu (SVA) retrotransposon insertion. The 5' truncated SVA element was inserted in a sense orientation in intron 11 of ITGB3, resulting in aberrant splicing of ITGB3 and significantly reducing ß3 protein content. Meanwhile, both the expression and transportation of ß3 were damaged by the ITGB3 c.2222G>A. Almost no αⅡb and ß3 expressions were detected on the patient's platelets surface. CONCLUSION: Novel compound heterozygous ITGB3 mutations were identified in the GT pedigree, resulting in defects of αⅡbß3 biosynthesis. This is the first report of SVA retrotransposon insertion in the genetic pathogenesis of GT. Our study highlights the importance of combining multiple high-throughput sequencing technologies for the molecular diagnosis of genetic disorders.

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.

RDA coupled with deep sequencing detects somatic SVA-retrotranspositions and mosaicism in the human brain.

Cells of the developing human brain are affected by the progressive acquisition of genetic and epigenetic alterations that have been reported to contribute to somatic mosaicism in the adult brain and are increasingly considered a possible cause of neurogenetic disorders. A recent work uncovered that the copy-paste transposable element (TE) LINE-1 (L1) is mobilized during brain development, and thus mobile non-autonomous TEs like AluY and SINE-VNTR-Alu (SVA) families can use L1 activity in trans, leading to de novo insertions that may influence the variability of neural cells at genetic and epigenetic levels. In contrast to SNPs and when considering substitutional sequence evolution, the presence or absence of TEs at orthologous loci represents highly informative clade markers that provide insights into the lineage relationships between neural cells and how the nervous system evolves in health and disease. SVAs, as the 'youngest' class of hominoid-specific retrotransposons preferentially found in gene- and GC-rich regions, are thought to differentially co-regulate nearby genes and exhibit a high mobility in the human germline. Therefore, we determined whether this is reflected in the somatic brain and used a subtractive and kinetic enrichment technique called representational difference analysis (RDA) coupled with deep sequencing to compare different brain regions with respect to de novo SINE-VNTR-Alu insertion patterns. As a result, we detected somatic de novo SVA integrations in all human brain regions analyzed, and the majority of de novo insertions can be attributed to lineages of telencephalon and metencephalon, since most of the examined integrations are unique to different brain regions under scrutiny. The SVA positions were used as presence/absence markers, forming informative sites that allowed us to create a maximum parsimony phylogeny of brain regions. Our results largely recapitulated the generally accepted evo-devo patterns and revealed chromosome-wide rates of de novo SVA reintegration targets and preferences for specific genomic regions, e.g., GC- and TE-rich regions as well as close proximity to genes that tend to fall into neural-specific Gene Ontology pathways. We concluded that de novo SVA insertions occur in the germline and somatic brain cells at similar target regions, suggesting that similar retrotransposition modes are effective in the germline and soma.

Mechanisms of disease-associated SINE-VNTR-Alus.

SINE-VNTR-Alus (SVAs) are the youngest retrotransposon family in the human genome. Their ongoing mobilization has generated genetic variation within the human population. At least 24 insertions to date, detailed in this review, have been associated with disease. The predominant mechanisms through which this occurs are alterations to normal splicing patterns, exonic insertions causing loss-of-function mutations, and large genomic deletions. Dissecting the functional impact of these SVAs and the mechanism through which they cause disease provides insight into the consequences of their presence in the genome and how these elements could influence phenotypes. Many of these disease-associated SVAs have been difficult to characterize and would not have been identified through routine analyses. However, the number identified has increased in recent years as DNA and RNA sequencing data became more widely available. Therefore, as the search for complex structural variation in disease continues, it is likely to yield further disease-causing SVA insertions.

Germline Chromothripsis Driven by L1-Mediated Retrotransposition and Alu/Alu Homologous Recombination.

Chromothripsis (CTH) is a phenomenon where multiple localized double-stranded DNA breaks result in complex genomic rearrangements. Although the DNA-repair mechanisms involved in CTH have been described, the mechanisms driving the localized "shattering" process remain unclear. High-throughput sequence analysis of a familial germline CTH revealed an inserted SVAE retrotransposon associated with a 110-kb deletion displaying hallmarks of L1-mediated retrotransposition. Our analysis suggests that the SVAE insertion did not occur prior to or after, but concurrent with the CTH event. We also observed L1-endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110-kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity facilitating DNA cleavage by catalytically active L1-endonuclease. Our data provide the first evidence that active and inactive human retrotransposons can serve as endogenous mutagens driving CTH in the germline.