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.

lncRNA BC200 is processed into a stable Alu monomer.

The non-coding RNA BC200 is elevated in human cancers and is implicated in translation regulation as well as cell survival and proliferation. Upon BC200 overexpression, we observed correlated expression of a second, smaller RNA species. This RNA is expressed endogenously and exhibits cell-type dependent variability relative to BC200. Aptamer tagged expression constructs confirmed that the RNA is a truncated form of BC200, and sequencing revealed a modal length of 120 nt, thus, we refer to the RNA fragment as BC120. We present methodology for accurate and specific detection of BC120 and establish that BC120 is expressed in several normal human tissues and is also elevated in ovarian cancer. BC120 exhibits remarkable stability relative to BC200 and is resistant to knock-down strategies that target the 3' unique sequence of BC200. Combined knock-down of BC200 and BC120 exhibits greater phenotypic impacts than knock-down of BC200 alone and overexpression of BC120 negatively impacts translation of a GFP reporter providing insight into a potential translational regulatory role for this RNA. The presence of a novel, truncated, and stable form of BC200 adds complexity to the investigation of this non-coding RNA that must be considered in future studies of BC200 and other related Alu RNAs.

Diagnostic and prognostic significance of ALU-based cell-free DNA in colorectal cancer: a systematic review and meta-analysis.

Introduction: Colorectal cancer (CRC) is a major global health concern. This study aimed to investigate the role of ALU-based cell-free DNA (cfDNA) in the diagnosis and prognosis of CRC. Methods: We selected relevant literature from PubMed, Scopus, Web of Science, EMBASE, and Science Direct databases based on strict inclusion and exclusion criteria. 17 eligible studies were included in the final analysis (13 studies for diagnostic and 4 studies for prognostic meta-analysis). The search covered relevant publications up to July 1, 2024. Results: The pooled sensitivity, specificity, and diagnostic odds ratios (DOR) of ALU-based cfDNA in CRC diagnosis were 0.81 (95% CI= [0.70, 0.89]), 0.90 (95% CI= [0.70, 0.96]), and 40.58 (95% CI= [17.87, 92.19]), respectively. The area under the ROC curve was 0.92 (95% CI= [0.89, 0.94]). Patients with higher concentrations of plasma/serum ALU-based cfDNA had poorer overall survival (OS) (pooled hazard ratio = 2.33 ([95% CI= [1.80, 3.03]). Conclusion: The current evidence supports the utility of circulating ALU as a promising non-invasive diagnostic and prognostic tool for CRC. Furthermore, as a potential biomarker, ALU-based cfDNA could play a significant role in clinical application. Clinical implications: The evidence suggests that circulating ALU-based cell-free DNA (cfDNA) holds promise as a non-invasive diagnostic and prognostic tool for colorectal cancer, potentially enhancing clinical decision-making. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier PROSPERO (CRD42023486369).

Uncovering hidden genetic variations: long-read sequencing reveals new insights into tuberous sclerosis complex.

Background: Tuberous sclerosis is a multi-system disorder caused by mutations in either TSC1 or TSC2. The majority of affected patients (85%-90%) have heterozygous variants, and a smaller number (around 5%) have mosaic variants. Despite using various techniques, some patients still have "no mutation identified" (NMI). Methods: We hypothesized that the causal variants of patients with NMI may be structural variants or deep intronic variants. To investigate this, we sequenced the DNA of 26 tuberous sclerosis patients with NMI using targeted long-read sequencing. Results: We identified likely pathogenic/pathogenic variants in 13 of the cases, of which 6 were large deletions, four were InDels, two were deep intronic variants, one had retrotransposon insertion in either TSC1 or TSC2, and one was complex rearrangement. Furthermore, there was a de novo Alu element insertion with a high suspicion of pathogenicity that was classified as a variant of unknown significance. Conclusion: Our findings expand the current knowledge of known pathogenic variants related to tuberous sclerosis, particularly uncovering mosaic complex structural variations and retrotransposon insertions that have not been previously reported in tuberous sclerosis. Our findings suggest a higher prevalence of mosaicism among tuberous sclerosis patients than previously recognized. Our results indicate that long-read sequencing is a valuable approach for tuberous sclerosis cases with no mutation identified (NMI).

Circulating tumor DNA in Egyptian women with breast Cancer: A marker for detection of primary cases and early prediction of recurrence.

Worldwide, female breast cancer (BC) has surpassed lung cancer as the most commonly diagnosed cancer. Early diagnosis of cancer recurrence can provide substantial benefits for BC patients who are at high risk of relapse. We aimed to investigate the role of ALU 247, ALU 115, cfDNA integrity index, CA15-3 and CEA as potential diagnostic markers in BC patients and as markers for early prediction of recurrence. Fifty BC patients (10 patients showed recurrence), 26 BBD patients and 22 healthy controls were included. Real-time q-PCR was used to measure the concentration of ALU 247 and ALU 115 in plasma then cfDNA integrity index was calculated. "ECLIA" was used to measure the concentration of CA15-3 and CEA in serum. Our results showed significant higher levels of ALU 247, ALU 115, CA15-3 and CEA in BC patients in comparison to healthy controls (P=0.02, 0.008, <0.001 and < 0.001 respectively). Also, cfDNA integrity index was higher in BC patients in comparison to healthy controls but statistically insignificance (p = 0.46). In recurrent BC patients; ALU 247, ALU 115, cfDNA integrity index, CA15-3 and CEA levels were higher compared to non-recurrent BC patients but with no statistic significant (p = 0.46, 0.59, 0.09, 0.85 and 0.84 respectively). This may result from the short period of follow up (1-2 years) and the relatively small sample size due to exclusion of patients with chronic diseases or inflammation as well as those who received therapy or post-surgery. By using the ROC curve, the sensitivity of ALU 247, ALU 115, CA15-3 and CEA for discriminating BC patients from BBD patients and healthy controls was 79 %, 79.2 %, 76.0 % and 88.0 % respectively. This study suggested that ALU 247, ALU 115, CA15-3 and CEA could be promising non-invasive markers of BC for diagnosis and early prediction of recurrence after validation in large-scale future studies.

A case study of Duchenne muscular dystrophy caused by Alu element insertion in DMD gene and analysis of its gray-hair symptoms.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive genetic disorder caused by mutations in the DMD gene, which leads to a deficiency of the dystrophin protein. The main mutation types of this gene include exon deletions and duplications, point mutations, and insertions. These mutations disrupt the normal expression of dystrophin, ultimately leading to the disease. In this study, we reported a case of DMD caused by an insertion mutation in exon 59 (E59) of the DMD gene. The affected child exhibited significant abnormalities in related biochemical markers, early symptoms of DMD, and multiple gray hair. His mother and sister were carriers with slightly abnormal biochemical markers. The mother had mild clinical symptoms, while the sister had no clinical symptoms. Other family members were genetically and physically normal. Sequencing and sequence alignment revealed that the inserted fragment was an Alu element from the AluYa5 subfamily. This insertion produced two stop codons and a polyadenylate (polyA) tail. To understand the impact of this insertion on the DMD gene and its association with clinical symptoms, exonic splicing enhancer (ESE) prediction indicated that the insertion did not affect the splicing of E59. Therefore, we speculated that the insertion sequence would be present in the mRNA sequence of the DMD gene. The two stop codons and polyA tail likely terminate translation, preventing the production of functional dystrophin protein, which may be the mechanism leading to DMD. In addition to typical DMD symptoms, the child also exhibited premature graying of hair. This study reports, for the first time, a case of DMD caused by the insertion of an Alu element into the coding region of the DMD gene. This finding provides clues for studying gene mutations induced by Alu sequence insertion and expands the understanding of DMD gene mutations.

An Evaluation of DNA Methylation Levels and Sleep in Relation to Hot Flashes: A Cross-Sectional Study.

Objectives: We aimed to evaluate the DNA methylation levels in perimenopausal and postmenopausal women, measured through Long Interspersed Element-1 (LINE-1) and Alu, and the sleep parameters in relation to the presence of hot flashes (HFs). Methods: This cross-sectional study included 30 peri- or postmenopausal women aged between 45 and 55. The menopausal status was determined according to STRAW + 10 criteria and all participants had a low cardiovascular disease (CVD) risk profile determined by Framingham risk score. The sample was divided into two groups based on the presence or absence of HFs documented in their medical history during their initial visit: Group 1 (n = 15) with HFs present and Group 2 (n = 15) with HFs absent. The patients had polysomnography test and HFs were recorded both by sternal skin conductance and self-report overnight. Genomic DNA was extracted from the women's blood and methylation status was analyzed by fluorescence-based real-time quantitative PCR. The quantified value of DNA methylation of a target gene was normalized by ß-actin. The primary outcome was the variation in methylation levels of LINE-1 and Alu and sleep parameters according to the presence of HFs. Results: LINE-1 and Alu methylation levels were higher in Group 1 (HFs present), although statistically non-significant. LINE-1 methylation levels were negatively correlated with age. Sleep efficiency was statistically significantly lower for women in Group 1 (HFs present) (74.66% ± 11.16% vs. 82.63% ± 7.31%; p = 0.03). The ratio of duration of awakening to total sleep time was statistically significantly higher in Group 1 (HFs present) (22.38% ± 9.99% vs. 15.07% ± 6.93, p = 0.03). Objectively recorded hot flashes were significantly higher in Group 1 (4.00 ± 3.21 vs. 1.47 ± 1.46, p = 0.03). None of the cases in Group 2 self-reported HF despite objectively recorded HFs during the polysomnography. The rate of hot flash associated with awakening was 41.4% in the whole sample. Conclusions: Women with a history of hot flashes exhibited lower sleep efficiency and higher awakening rates. Although a history of experiencing hot flashes was associated with higher LINE-1 and Alu methylation levels, no statistical significance was found. Further studies are needed to clarify this association. This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa. Project number: TTU-2021-35629.

Evaluating circulating cell-free DNA and DNA integrity index as biomarkers in non-small cell lung cancer.

BACKGROUND: Analysis of free DNA molecules shed from tumour cells in plasma of patients referred as circulating tumour DNA (ctDNA) with reference to physiological circulating cell-free DNA (cfDNA) is nowadays exploited as liquid biopsy and is considered a new emerging promising biomarker for diagnosis, selection of proper treatment, and prognosis of cancer. DNA integrity index (DII) is assessed by calculating the ratio between the concentration of long cfDNA strands released from tumour cells (ALU247) and the short strands released from normal cells (ALU115). The aim of the current study was to evaluate DII as a potential diagnostic and prognostic biomarker of NSCLC. METHODS: Our study included 48 NSCLC patients diagnosed as primary NSCLC before starting treatment, 30 COPD patients diagnosed clinically, radiologically, and subjected to chest high-resolution computerized tomography, and 40 healthy controls. cfDNA concentration and DII were measured by quantitative real-time polymerase chain reaction (qPCR). RESULTS: ALU115, ALU247, and DII were significantly higher in NSCLC compared to COPD patients (p < 0.0001) and controls (p < 0.0001) and in COPD patients compared to control subjects (p < 0.0001). DII positively correlated with the stage of tumour (p = 0.01), tumour metastasis (p = 0.004), and with adenocarcinoma compared to other histopathological types (p = 0.02). To evaluate clinical utility of DII in NSCLC, ROC curve analysis demonstrated an AUC of 0.91 at a cut-off value of 0.44 with total accuracy = 85.6%, sensitivity = 90%, specificity = 83%, PPV = 78.1%, and NPV = 92.1%. CONCLUSION: cfDNA and DII represent a promising diagnostic and prognostic tool in NSCLC. This type of noninvasive liquid biopsy revealed its chance in the screening, early diagnosis, and monitoring of NSCLC.

Germline PTCH1: c.361_362insAlu alteration identified by comprehensive exome and RNA sequencing in a patient with Gorlin syndrome.

Gorlin syndrome can be caused by pathogenic/likely pathogenic (P/LP) variants in the tumor suppressor gene PTCH1 (9q22.1-q31), which encodes the receptor for the sonic hedgehog (SHH) ligand. We present a 12-month-old boy clinically diagnosed with Gorlin syndrome who was found to have significantly delayed development, palmar pitting, palmar and plantar keratosis, short hands, frontal bossing, coarse face, hypertelorism, a bifid rib, misaligned and missing teeth, and SHH-activated medulloblastoma. Genetic testing, including a pediatric cancer panel and genome sequencing with peripheral blood, failed to identify any P/LP variants in PTCH1. Paired tumor/normal exome sequencing was performed, which identified a germline NM_000264.5 (PTCH1): c.361_362ins? alteration through manual review of sequencing reads. Clinical RNA sequencing further demonstrated an Alu insertion at this region (PTCH1: c.361_362insAlu), providing molecular confirmation of Gorlin syndrome. This finding exemplifies a unique mechanism for PTCH1 disruption in the germline and highlights the importance of comprehensive analysis, including manual review of DNA sequencing reads and the utility of RNA analysis to detect variant types which may not be identified by routine genetic screening techniques.

Measuring cfDNA integrity as a biomarker for predicting neoadjuvant chemotherapy response in breast cancer patients: a pilot study.

PURPOSE: Circulating cell-free DNA (cfDNA) is a promising biomarker for predicting treatment response and disease outcomes in Breast Cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC). To determine if cfDNA originates from tumors, matching tumor and cfDNA gene mutations are necessary, often requiring tumor DNA sequencing. We assessed plasma cfDNA integrity by measuring concentrations and ratios of larger-to-smaller Alu DNA fractions as a potential biomarker, eliminating the need for prior tumor sequencing. METHODS: We included patients with localized and/or locally advanced BC receiving standard NAC alone or in combination with immunotherapy and/or anti-HER2 targeted therapy. Blood samples were collected before treatment, every 2 weeks during treatment, and before surgery. RESULTS: Of the 38 evaluated patients, only 28 completed the protocol and underwent surgery after NAC. Seven patients (25%) achieved a pathologic complete response (pCR). We found that cfDNA integrity (cfDNAI) levels at 15 days after starting NAC were significantly higher in patients who achieved pCR (p = 0.045) and correlated significantly with Disease-Free Survival (DFS) in univariate analysis (p = 0.0371). CONCLUSIONS: Evaluation of cfDNAI 2 weeks after NAC initiation appears to be an early biomarker for tumor pCR and DFS. Measuring Alu fragments of different lengths may replace techniques requiring prior tumor sequencing to measure ctDNA, reducing costs and complexity of cfDNA serial measurements in BC patients undergoing NAC.

hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Nuclear aggregates of NONO/SFPQ and A-to-I-edited RNA in Parkinson's disease and dementia with Lewy bodies.

Neurodegenerative diseases are commonly classified as proteinopathies that are defined by the aggregation of a specific protein. Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are classified as synucleinopathies since α-synuclein (α-syn)-containing inclusions histopathologically define these diseases. Unbiased biochemical analysis of PD and DLB patient material unexpectedly revealed novel pathological inclusions in the nucleus comprising adenosine-to-inosine (A-to-I)-edited mRNAs and NONO and SFPQ proteins. These inclusions showed no colocalization with Lewy bodies and accumulated at levels comparable to α-syn. NONO and SFPQ aggregates reduced the expression of the editing inhibitor ADAR3, increasing A-to-I editing mainly within human-specific, Alu-repeat regions of axon, synaptic, and mitochondrial transcripts. Inosine-containing transcripts aberrantly accumulated in the nucleus, bound tighter to recombinant purified SFPQ in vitro, and potentiated SFPQ aggregation in human dopamine neurons, resulting in a self-propagating pathological state. Our data offer new insight into the inclusion composition and pathophysiology of PD and DLB.

Long-read sequencing improves the genetic diagnosis of retinitis pigmentosa by identifying an Alu retrotransposon insertion in the EYS gene.

BACKGROUND: Biallelic variants in EYS are the major cause of autosomal recessive retinitis pigmentosa (arRP) in certain populations, a clinically and genetically heterogeneous disease that may lead to legal blindness. EYS is one of the largest genes (~ 2 Mb) expressed in the retina, in which structural variants (SVs) represent a common cause of disease. However, their identification using short-read sequencing (SRS) is not always feasible. Here, we conducted targeted long-read sequencing (T-LRS) using adaptive sampling of EYS on the MinION sequencing platform (Oxford Nanopore Technologies) to definitively diagnose an arRP family, whose affected individuals (n = 3) carried the heterozygous pathogenic deletion of exons 32-33 in the EYS gene. As this was a recurrent variant identified in three additional families in our cohort, we also aimed to characterize the known deletion at the nucleotide level to assess a possible founder effect. RESULTS: T-LRS in family A unveiled a heterozygous AluYa5 insertion in the coding exon 43 of EYS (chr6(GRCh37):g.64430524_64430525ins352), which segregated with the disease in compound heterozygosity with the previously identified deletion. Visual inspection of previous SRS alignments using IGV revealed several reads containing soft-clipped bases, accompanied by a slight drop in coverage at the Alu insertion site. This prompted us to develop a simplified program using grep command to investigate the recurrence of this variant in our cohort from SRS data. Moreover, LRS also allowed the characterization of the CNV as a ~ 56.4kb deletion spanning exons 32-33 of EYS (chr6(GRCh37):g.64764235_64820592del). The results of further characterization by Sanger sequencing and linkage analysis in the four families were consistent with a founder variant. CONCLUSIONS: To our knowledge, this is the first report of a mobile element insertion into the coding sequence of EYS, as a likely cause of arRP in a family. Our study highlights the value of LRS technology in characterizing and identifying hidden pathogenic SVs, such as retrotransposon insertions, whose contribution to the etiopathogenesis of rare diseases may be underestimated.

Biallelic NDUFA4 Deletion Causes Mitochondrial Complex IV Deficiency in a Patient with Leigh Syndrome.

Oxidative phosphorylation involves a complex multi-enzymatic mitochondrial machinery critical for proper functioning of the cell, and defects herein cause a wide range of diseases called "primary mitochondrial disorders" (PMDs). Mutations in about 400 nuclear and 37 mitochondrial genes have been documented to cause PMDs, which have an estimated birth prevalence of 1:5000. Here, we describe a 4-year-old female presenting from early childhood with psychomotor delay and white matter signal changes affecting several brain regions, including the brainstem, in addition to lactic and phytanic acidosis, compatible with Leigh syndrome, a genetically heterogeneous subgroup of PMDs. Whole genome sequencing of the family trio identified a homozygous 12.9 Kb deletion, entirely overlapping the NDUFA4 gene. Sanger sequencing of the breakpoints revealed that the genomic rearrangement was likely triggered by Alu elements flanking the gene. NDUFA4 encodes for a subunit of the respiratory chain Complex IV, whose activity was significantly reduced in the patient's fibroblasts. In one family, dysfunction of NDUFA4 was previously documented as causing mitochondrial Complex IV deficiency nuclear type 21 (MC4DN21, OMIM 619065), a relatively mild form of Leigh syndrome. Our finding confirms the loss of NDUFA4 function as an ultra-rare cause of Complex IV defect, clinically presenting as Leigh syndrome.

Maternal non-compliance with recommended folic acid supplement use alters global DNA methylation in cord blood of newborns: A cohort study.

BACKGROUND & AIMS: Prenatal folate exposure may alter epigenetic marks in the offspring. We aimed to evaluate associations between prenatal exposure to folic acid (FA) in preconception and in utero with cord blood DNA methylation in long interspersed nuclear element 1 (LINE-1) and Alu short interspersed nuclear elements (SINEs) as markers of global DNA methylation levels. METHODS: Data come from 325 mother-child pairs participating in the Nutrition in Early Life and Asthma (NELA) birth cohort (2015-2018). Pregnant women were asked about supplement use, including brand name and dose, one month before pregnancy (preconception) and through the trimesters of pregnancy. Maternal dietary folate intake was assessed using a validated food frequency questionnaire with additional questions for FA supplement use. Folate serum levels were measured in mothers at 24 weeks of gestation and in cord blood of newborns. DNA methylation was quantitatively assessed by bisulfite pyrosequencing on 5 LINE-1 and 3 Alu different elements. Associations were estimated using multivariable linear regression models. RESULTS: A reduction in methylation levels of LINE-1 in newborns was associated with the use of FA supplements below the recommended doses (<400 ug/day) during preconception (-0.50; 95% CI: -0.91, -0.09; P = 0.016), and from preconception up to 12 weeks of gestation (-0.48; 95% CI: -0.88, -0.08; P = 0.018). Maternal use of FA supplements above the tolerable upper intake level of 1000 ug/day from preconception until 12 weeks of gestation was also related to lower methylation in LINE-1 at birth (-0.77; 95% CI: -1.52, -0.02; P = 0.044). Neither FA supplement use after 12 weeks of gestation nor maternal total folate intake (diet plus supplements) were associated with global DNA methylation levels at birth. CONCLUSIONS: Maternal non-compliance with the use of FA supplement recommendations from preconception up to 12 weeks of gestation reduces offspring global DNA methylation levels at birth.

Novel Alu insertion in the ZEB2 gene causing Mowat-Wilson syndrome.

Alu elements are short, interspersed elements located throughout the genome, playing a role in human diversity, and occasionally causing genetic diseases. Here, we report a novel Alu insertion causing Mowat-Wilson syndrome, a rare neurodevelopmental disorder, in an 8-year-old boy displaying the typical clinical features for Mowat-Wilson syndrome. The variant was not initially detected in genome sequencing data, but through deep phenotyping, which pointed to only one plausible candidate gene, manual inspection of genome sequencing alignment data enabled us to identify a de novo heterozygous Alu insertion in exon 8 of the ZEB2 gene. Nanopore long-read sequencing confirmed the Alu insertion, leading to the formation of a premature stop codon and likely haploinsufficiency of ZEB2. This underscores the importance of deep phenotyping and mobile element insertion analysis in uncovering genetic causes of monogenic disorders as these elements might be overlooked in standard next-generation sequencing protocols.

Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

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.

Polyamine Dysregulation and Nucleolar Disruption in Alzheimer's Disease.

A hypothesis of Alzheimer's disease etiology is proposed describing how cellular stress induces excessive polyamine synthesis and recycling which can disrupt nucleoli. Polyamines are essential in nucleolar functions, such as RNA folding and ribonucleoprotein assembly. Changes in the nucleolar pool of anionic RNA and cationic polyamines acting as counterions can cause significant nucleolar dynamics. Polyamine synthesis reduces S-adenosylmethionine which, at low levels, triggers tau phosphorylation. Also, polyamine recycling reduces acetyl-CoA needed for acetylcholine, which is low in Alzheimer's disease. Extraordinary nucleolar expansion and/or contraction can disrupt epigenetic control in peri-nucleolar chromatin, such as chromosome 14 with the presenilin-1 gene; chromosome 21 with the amyloid precursor protein gene; chromosome 17 with the tau gene; chromosome 19 with the APOE4 gene; and the inactive X chromosome (Xi; aka "nucleolar satellite") with normally silent spermine synthase (polyamine synthesis) and spermidine/spermine-N1-acetyltransferase (polyamine recycling) alleles. Chromosomes 17, 19 and the Xi have high concentrations of Alu elements which can be transcribed by RNA polymerase III if positioned nucleosomes are displaced from the Alu elements. A sudden flood of Alu RNA transcripts can competitively bind nucleolin which is usually bound to Alu sequences in structural RNAs that stabilize the nucleolar heterochromatic shell. This Alu competition leads to loss of nucleolar integrity with leaking of nucleolar polyamines that cause aggregation of phosphorylated tau. The hypothesis was developed with key word searches (e.g., PubMed) using relevant terms (e.g., Alzheimer's, lupus, nucleolin) based on a systems biology approach and exploring autoimmune disease tautology, gaining synergistic insights from other diseases.

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.