Abi Pfaff

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder presenting progressive weakness of the bulbar and extremity muscles, leading to a wide-ranging clinical phenotype. More than 30 genes have been associated to genetically inherited ALS yet, approximately 85%–90% of ALS cases are sporadic. Short tandem repeats expansions, have recently been found in clinically diagnosed ALS patients and are currently investigated as potential genetic biomarkers. In this paper we compare the investigation of pathological tandem repeat expansions on a group of ALS patients by comparing the standard short-read sequencing (SRS) technique with a long-read-sequencing (LRS) method which has recently become more accessible. Blood samples from 47 sporadic ALS cases were subjected to SRS by Illumina Whole Genome Sequencing. The genome-wide tandem repeat expansions were genotyped using GangSTR, while wANNOVAR was used for variant annotation. Uncertain cases were further explored using LRS. SRS identified pathological expansions in HTT , ATXN2 , and CACNA1A genes in one patient, which were not confirmed with LRS. The latter identified large tandem repeat expansions in the C9orf72 gene of one patient that were missed by SRS. Our findings suggest that LRS should be preferred to SRS for accurate identification of pathological tandem repeat expansions.

An individual’s genetics contributes to their risk of developing amyotrophic lateral sclerosis (ALS); however, there is still a large proportion of the heritability of ALS to be understood. Part of this missing heritability may lie in complex variants, such as the long interspersed element 1 (L1) retrotransposon, which have yet to be evaluated. The majority of L1 insertions in the human genome are no longer able to retrotranspose, but to date 279 retrotransposition-competent (RC) L1s have been reported. Many RC-L1s are polymorphic for their presence/absence; therefore, each individual will have a different number and complement of RC-L1s. These elements have been hypothesized to be involved in disease processes by multiple mechanisms such as somatic mutation by retrotransposition, the triggering of neuroinflammation and DNA damage. We hypothesize that L1s may influence disease development either through their effects on endogenous genes or through the properties that enable them to retrotranspose. Whole genome sequencing data from the New York Genome Center ALS consortium were used to characterize L1 variation identifying 2,803 polymorphic L1 elements and association analysis was performed in European individuals (ALS/ALS with other neurological disorder (ALSND) n = 2,653, controls n = 320). There were no individual L1 elements associated with disease, but we did identify a significant increase in the number of RC-L1s in ALS/ALSND genomes (p = 0.01) and the presence of ≥46 RC-L1s showed the most significant association (OR = 1.09 (1.02–1.16), p = 0.01) with disease. Analysis of individual L1s and their association with age at onset and survival identified one L1 whose presence was significantly associated with a lower age at onset (52.7 years) compared to homozygous absent individuals (59.2 years) (padj = 0.009). Our study has identified novel genetic factors for both disease risk and age at onset in ALS providing further evidence for the role of L1 retrotransposons in neurodegenerative diseases.

Background/Objectives: The highly polymorphic Major Histocompatibility Complex (MHC) genomic region, located on the short arm of chromosome 6, is implicated genetically in Parkinson’s disease (PD), a progressive neurodegenerative disorder with motor and non-motor symptoms. Previously, we reported significant associations between SINE-VNTR-Alu (SVA) expression quantitative trait loci (eQTLs) and Human Leucocyte Antigen (HLA) class I genotypes in PD. In this study, we aimed to evaluate SVA associations and their regulatory effects on transposable element (TE) transcription in the MHC class I region.

Methods: Transcriptome data from the peripheral blood cells of 1530 individuals in the Parkinson’s Progression Markers Initiative (PPMI) cohort were reanalyzed for TE and gene expression using publicly available bioinformatics tools, including Salmon and Matrix-eQTL.

Results: Four structurally polymorphic SVAs regulated the transcription of 18 distinct clusters of 235 TE loci, comprising LINEs (33%), SINEs (19%), LTRs (35%), and ancient transposon DNA elements (12%) located near HLA genes. The transcribed TEs were predominantly short, with an average length of 445 nucleotides. The regulatory effects of these SVAs varied significantly in terms of TE types, numbers, and transcriptional activation or repression. The SVA-regulated TE RNAs in blood cells appear to function as enhancer-like elements, differentially influencing the expression of HLA class I genes, non-HLA genes, and noncoding RNAs.

Conclusions: These findings highlight the roles of SVAs and their associated TEs in the complex regulatory networks governing coding and noncoding gene expression in the MHC class I region, with potential implications for immune function and disease susceptibility.

Blood-based biomarkers for motor neuron disease are needed for better diagnosis, progression prediction, and clinical trial monitoring. We used whole blood-derived total RNA and performed whole transcriptome analysis to compare the gene expression profiles in (motor neurone disease) MND patients to the control subjects. We compared 42 MND patients to 42 aged and sex-matched healthy controls and described the whole transcriptome profile characteristic for MND. In addition to the formal differential analysis, we performed functional annotation of the genomics data and identified the molecular pathways that are differentially regulated in MND patients. We identified 12,972 genes differentially expressed in the blood of MND patients compared to age and sex-matched controls. Functional genomic annotation identified activation of the pathways related to neurodegeneration, RNA transcription, RNA splicing and extracellular matrix reorganisation. Blood-based whole transcriptomic analysis can reliably differentiate MND patients from controls and can provide useful information for the clinical management of the disease and clinical trials.

Background
Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD), effecting ∼ 6 million individuals world-wide. Although clinically characterised by motor dysfunction, psychiatric symptoms and cognitive impairment are observed in 30-50% of PD patients. These non-motor symptoms have significant impact on patient well-being and are associated with a faster increase in disease severity over time. PD is itself significantly heritable and polygenic, with 90 independent risk loci identified in genome-wide analysis. As such, genetically informed approaches such as polygenic risk scores (PRS) may help us better understand the underlying biological contribution to psychiatric and cognitive outcomes in PD. Transcriptional risk scores (TRS) – the sum of an individuals observed gene expression weighted by their Z-score from transcriptome-wide association analysis (TWAS) – offer a complementary approach to PRS, having shown predictive utility in previous studies of AD, amyotrophic lateral sclerosis (ALS) and Crohn's disease.

Methods
Following TWAS, we calculated PRS and TRS for AD, depression (DEP), PD and schizophrenia (SCZ) in the Parkinson's Progression Markers Initiative (PPMI) (Ncases = 431; Ncontrols = 161) and tested their association with PD-related (case/control status, age of onset, overall PD severity), psychiatric (depression, anxiety, apathy and hallucinations) and cognitive outcomes (overall cognitive impairment, working memory, processing speed, verbal learning, visuospatial ability). Associations with these outcomes were assessed at baseline and longitudinally, extracting individual random slopes for each outcome across up to ten years of follow-up using mixed-effects models. Analyses were restricted to cases-only except for case/control status. An FDR correction was applied to results correcting for 176 tests (pFDR ≤ 0.05).

Results
Both PRS and TRS for PD were significantly associated with PD case/control status, although the association was stronger for the PRS (R2 = 0.17 vs. 0.015, pFDR = 7.40e-16 vs. 0.038). Higher DEP-PRS was significantly associated with higher anxiety (pFDR = 0.031) and depression (pFDR = 0.046) in PD cases at baseline, while higher PD and AD TRS were associated with hallucinations (pFDR = 0.046 and 0.031 respectively). Higher SCZ-PRS was associated with a faster increase in PD severity over time (pFDR = 0.031). No associations were observed for the longitudinal slopes of psychiatric outcomes. However, significant associations were identified between higher levels of all TRS and faster cognitive decline across multiple domains, including overall cognitive impairment (pFDR range: 0.033-0.001). No such associations were observed for the PRS, highlighting the utility of TRS in risk prediction.

Discussion
Findings suggest that transcriptional risk scores can be applied to help us better understand the way disease-related biological risk influences psychiatric and cognitive outcomes in PD. Forthcoming analyses will seek to disaggregate transcriptional scores to identify significant gene-sets driving associations, with a particular focus on TRS associated with cognitive decline.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.

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.

SINE-VNTR-Alu (SVA) retrotransposons can regulate expression quantitative trait loci (eQTL) of coding and noncoding genes including transposable elements (TEs) distributed throughout the human genome. Previously, we reported that expressed SVAs and human leucocyte antigen (HLA) class II genotypes on chromosome 6 were associated significantly with Parkinson's disease (PD). Here, our aim was to follow-up our previous study and evaluate the SVA associations and their regulatory effects on the transcription of TEs within the HLA class II genomic region. We reanalyzed the transcriptome data of peripheral blood cells from the Parkinson's Progression Markers Initiative (PPMI) for 1530 subjects for TE and gene RNAs with publicly available computing packages. Four structurally polymorphic SVAs regulate the transcription of 20 distinct clusters of 235 TE loci represented by LINES (37%), SINES (28%), LTR/ERVs (23%), and ancient transposon DNA elements (12%) that are located in close proximity to HLA genes. The transcribed TEs were mostly short length, with an average size of 389 nucleotides. The numbers, types and profiles of positive and negative regulation of TE transcription varied markedly between the four regulatory SVAs. The expressed SVA and TE RNAs in blood cells appear to be enhancer-like elements that are coordinated differentially in the regulation of HLA class II genes. Future work on the mechanisms underlying their regulation and potential impact is essential for elucidating their roles in normal cellular processes and disease pathogenesis.

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.
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
and the
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.
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
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
and -
protective alleles (PD v HC), the
risk (HC v Prodrome) or protective allele (PD v Prodrome), the
and -
risk alleles (SWEDD v HC), and the
present genotype (PD v HC) at a 5% homozygous insertion frequency near
, were significant (
) after Bonferroni corrections. The homologous
insertion significantly decreased the transcription levels of
and
in the PPMI cohort and its presence as a homozygous genotype is a risk factor (
) for PD. The most frequent
insertion haplotype in the PPMI cohort was
(3.7%). Although
was the most frequent HLA 5-loci phased-haplotype (n, 76) in the PPMI cohort, the
insertion was present in only six of them (8%).
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.