Role Of TOMM40'523' Polymorphism in Disease Risk And Age At Symptom Onset in Two Independent Parkinson's Disease Cohorts

Abstract

Abnormal mitochondrial function is a key process in the pathogenesis of Parkinson’s disease (PD). The central pore-forming protein TOM40 of the mitochondria is encoded by the translocase of the outer mitochondrial membrane 40 homolog gene (TOMM40). The highly variant ‘523’ poly-T repeat is associated with age-related cognitive decline and age of onset in Alzheimer’s disease, but whether it plays a role in modifying the risk or clinical course of PD is yet to be elucidated. The TOMM40 ‘523’ allele length was determined in 634 people with PD and 422 healthy controls from an Australian cohort and the Parkinson’s Progression Markers Initiative (PPMI) cohort, using polymerase chain reaction or whole genome sequencing analysis. Genotype and allele frequencies of TOMM40 ‘523’ and APOE ε did not differ significantly between the cohorts. Analyses revealed TOMM40 ‘523’ allele groups were not associated with disease risk while considering APOE ε genotype. Regression analyses revealed the TOMM40 S/S genotype was associated with a significantly later age of symptom onset in the PPMI PD cohort, but not after correction for covariates, or in the Australian cohort. Whilst variation in the TOMM40 ‘523’ polymorphism was not associated with PD risk, the possibility that it may be a modifying factor for the age of symptom onset warrants further investigation in other PD populations.

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Introduction

Parkinson’s disease (PD) is increasingly known as a multifaceted neurodegenerative disorder with a heterogeneous and burdensome symptom presentation and progression. Abnormal or deficient mitochondrial functioning is widely implicated as a key process in the selective neuronal death and pathogenesis of PD. Mitochondrial dysfunction, resulting in a loss of electron transport chain (ETC) efficiency or decline in ATP-synthesising capacity, appears to elicit dopaminergic cell death via several mechanisms, including reactive oxygen species(ROS)—generation, impaired ATP production, and disrupted calcium homeostasis. The pivotal role of mitochondrial dysfunction in PD is supported by several parkinsonism-causing toxins, genetic mutations, and retrotransposon insertions, which specifically impair mitochondrial function. Insights from these toxins and mutations imply that mitochondrial dysfunction in the pathogenesis of PD can arise from a wide array of biological processes, such as bioenergetic disturbances, nuclear and mitochondrial DNA mutations, impaired fusion and fission, defective mitophagy, abnormal morphology, and size. For example, a significant cause of mitochondrial dysfunction in PD is the inhibition of mitochondrial complex I, an ETC defect that leads to severe oxidative stress and ROS- and caspase-mediated dopaminergic cell death. Notably, the latter impairment is a major pathological feature of PD induced by familial PTEN-induced kinase 1 (PINK1), alpha-synuclein (SNCA), and Daisuke-Junko-1 (DJ-1) gene mutations or the toxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). Thus, mitochondrial dysfunction is thought to cause neuronal stress and degeneration, eventuating in this neurodegenerative disease, PD.

The translocase of the outer mitochondrial membrane 40 homologs (TOMM40) gene encodes the pore-forming subunit (TOM40) of the protein-transport channels in the mitochondrial outer membrane, thus playing a fundamental role in mitochondrial functioning. Te TOM40 protein facilitates the import of approximately 1500 externally synthesized proteins and peptides into the mitochondria and plays a key role in the mitophagy degradation pathway. Altered or abnormally functioning TOM40, mediated by genetic changes or atypical protein expression, is thought to contribute to mitochondrial dysfunction and protein accumulation in Alzheimer’s disease (AD) and PD. This may occur through different mechanisms: (1) mitochondrial import impairment, which may prevent essential proteins and peptides from reaching their designated mitochondrial targets, or allow unwanted and mutant proteins to aggregate in the mitochondria; or (2) mitophagy disruption, which may enable damaged and malfunctioning mitochondria to accumulate. Given the evidence for mitochondrial dysfunction in PD, genetic variants affecting TOMM40 expression may have risk and disease-modifying effects.

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Downstream of TOMM40 is apolipoprotein E (APOE), which is in a region of strong linkage disequilibrium (LD), or non-random association, with TOMM40 variation. Though the extent to which common variants in TOMM40 can have APOE-independent effects on disease risk or disease-modifying effects remains uncertain, polymorphisms in TOMM40 have been independently associated with a range of primarily cognition-based neurodegenerative diseases, including AD, frontotemporal dementia, and dementia with Lewy bodies, as well as non-pathological aging. One of these variants is situated in intron 6 of TOMM40 and is a homopolymer of repeating thymine (T) nucleotides known as rs10524523 or ‘523’. The ‘523’ variant is polymorphic, with lengths that vary from 11 to 39 T residues in Caucasians. To better categorize and describe this diverse structural variant, three allelic groups for the homopolymer have been established previously, according to the number of constituent T residues: ‘Short’ (S, T≤19), ‘Long’ (L, 20≤T≤29) and ‘Very Long’ (T≥30). Although the functional effects of the ‘523’ variation are mostly unknown, increasing length of the poly-T repeat is thought to increase TOMM40 expression. Therefore, the effect of the TOMM40 ‘523’ variant and altered TOM40 protein levels may contribute to mitochondrial dysfunction in PD. While a vast number of published studies report conflicting roles of the various TOMM40 ‘523’ variant lengths in AD and aging populations, there have only been two previous studies within PD cohorts with different findings. The first was a study of a relatively large Polish PD cohort which found no significant associations between TOMM40 ‘523’ and PD risk or the age of onset. Subsequently, a significant overrepresentation of the L/VL ‘523’ genotype was reported in a Swedish PD cohort compared to healthy controls, though it should be noted that these findings are yet to be published in full. Thus, with conflicting findings in these two European cohorts, further investigation in other populations is required.

Due to the essential role of TOMM40 in mitochondrial import and mitophagy, both plausible sources of PDcausing mitochondrial dysfunction, the ‘523’ variant in TOMM40 is, therefore, a potential risk factor and disease modifier in PD. This study investigated the distribution of ‘523’ alleles and their association with disease risk and age of symptom onset in two independent PD cohorts, one Australian and one international (the Parkinson’s Progression Markers Initiative, PPMI), using PCR-based and whole genome sequencing approaches.

Discussion

The current study aimed to investigate the TOMM40 ‘523’ structural variant as a potential PD risk factor and modifier of the age at symptom onset. The distribution of ‘523’ poly-T alleles did not vary between PwP and healthy controls, or in the two independent cohorts examined. The similarity in ‘523’ allelic distribution between the PPMI and Australian cohorts is worth noting as this study utilized two different approaches in the calling of the TOMM40 ‘523’ variant, establishing a PCR-based and a WGS-based assay for use in the Australian and PPMI cohorts, respectively. Assay development for the TOMM40 ‘523’ variant is generally considered to be difficult, as poly-T variants are challenging to sequence. Despite optimization within this study, PCR stuttering was observed in the PCR-based assay similar to previous reports, which is a standard complication when amplifying repetitive genomic sequences, and highlights the need for further optimization of the assay. By comparison, calling from WGS files revealed a similar distribution as reported previously. While it has been presented as having fewer challenges in optimization and sequencing, calling of ‘523’ variants was an arduous process and the aforementioned study has stated that the correlation between ‘523’ calling by WGS and PCR-based methods decreases with increasing size of the ‘523’ allele.

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The present study then examined whether the TOMM40 ‘523’ variant is implicated in PD risk and found no association between the carriage of allelic variants and the risk of developing PD. When binned, genotypic and allelic frequencies appeared to be similar between controls and PwP in both independent cohorts, and when the cohorts were combined. This agrees with previous work in a Polish PD cohort which showed no association of ‘523’ alleles, genotypes, or haplotypes with the risk of PD or the age of symptom onset. In contrast, in a Swedish population, a higher frequency of the L/VL genotype was observed in PD patients compared to controls, though these findings are yet to be published in full. Given that these studies were conducted in different ethnic groups, and that ‘523’ allele frequencies are ethnic-specific, the contradictory findings in the studies to date may indicate that ‘523’ is a risk factor in some populations, but not in others. As this is only the third study to investigate the role of TOMM40 ‘523’ in PD risk, further studies should be performed in other ethnically diverse populations.

As mitochondrial involvement in PD is thought to be a key contributor to neuronal dysfunction and degeneration, it is plausible that a genetic variant that modulates mitochondrial function could modify the age of symptom onset. Furthermore, AD and PD share several clinical, pathological, and molecular features including toxic protein accumulation and mitochondrial dysfunction in the form of respiratory chain defects, oxidative stress, mitochondrial DNA damage, and morphological abnormalities. Previous studies have implicated TOMM40 ‘523’ length in the age of onset of AD, particularly in carriers of the APOE ε3 allele, but mixed findings exist. For instance, although Roses et al. reported that longer ‘523’ allele lengths were associated with the risk of AD and earlier age of onset, the initial findings were not replicated in other populations. Such varied findings may be a result of varied methodologies and varied considerations of the influence of the APOE ε locus, which is well-established as the strongest genetic predictor of AD. Most studies to date report no association of APOE ε variation with susceptibility to PD, as was also the case in the current study. While the present findings did not show any positive evidence of interactive effects between TOMM40 ‘523’ and APOE ε genotypes about PD risk, they do suggest that the two loci may have small independent and opposing effects on determining the age of onset of the symptoms. While initial regression analyses in the PPMI cohort showed that the age of onset was significantly delayed by the carriage of the S/S ‘523’ genotype, this was not replicated after correction for covariates, or in the Australian cohort. However, this difference may be caused by a country- or geographical-specific effect, as seen in other genetic studies. It is worth noting that only one prior study has examined this association, though it is not clear whether this study completed a rigorous analysis of the interactive effects of TOMM40 and APOE, as was conducted in the current study. As such, further in-depth analysis in larger PD cohorts is required to determine the significance of the present findings.

Currently, the functional effects of variation in the ‘523’ allele length are poorly understood due to a scarcity of research in this area. While several studies have suggested that the VL allele increases TOMM40 mRNA expression and the S allele represses expression, others have found no significant differences in mRNA levels between S and VL variants. A recent study demonstrated that overexpression of TOMM40 in vitro was associated with greater mitochondrial membrane potentials, respiratory rates, spare respiratory capacities, ATP levels, amyloid-beta resistance, and protein uptake. On the other hand, another study observed a correlation between TOM40 protein deficits and enhanced oxidative stress, reduced ATP production, and abnormal complex I protein concentrations in the brains of PD patients and alpha-synuclein overexpressing murine models. As the literature currently stands, further elucidation of the biological consequences of up or down-regulated TOMM40 expression is required to give insight into its potential role in PD risk and disease modification.

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Conclusion

Overall, this study aimed to investigate the risk and disease-modifying role of the TOMM40 ‘523’ variant in two independent PD cohorts. TOMM40 plays an essential role in mitochondrial import and mitophagy, and the ‘523’ polymorphism has been associated with the age of onset of AD and with age-related cognitive decline. While this study clarifies that TOMM40 ‘523’ is not in itself a predictor of PD risk, it raises the possibility that, as in AD, it may be a genetic marker for the age of symptom onset in PD. Whilst not conclusive, our findings in the large international PPMI cohort suggest that carriage of the S/S ‘523’ genotype may be protective in terms of delaying the age of symptom onset, and may warrant further investigation in other populations. Importantly, the effect of co-carriage of the APOE ε4 allele, which appears to have an opposing effect on the age of onset, must be considered in future studies. Though not significant in modulating the risk of PD, future studies should consider the possible role of TOMM40 ‘523’ as a determinant of the age of symptom onset, and symptom trajectory. Given a phase 3 clinical trial of a therapeutic for the prevention and delay of onset of AD was recently conducted involving participants stratified by TOMM40 ‘523’46, the findings reported herein are noteworthy and may allow the design of symptom-focused studies in PD for much-needed improvements in patient outcomes and care.

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Megan C. Bakeberg 1,2, Madison E. Hoes 1 , Anastazja M.Gorecki 1,3, FrancesTheunissen 1,4, Abigail L. Pfaf 1,4, Jade E. Kenna 1,2, Kai Plunkett 1 , Sulev Kõks 1,4, P.AnthonyAkkari 1,2,4, Frank L. Mastaglia 1,2,4 & Ryan S.Anderton 1,2,5.

1. Perron Institute for Neurological and Translational Science, Nedlands, WA, Australia.

2. Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Nedlands, WA, Australia.

3. School of Biological Sciences, University of Western Australia, Crawley, WA, Australia.

4. The Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Murdoch, WA, Australia.

5. Institute for Health Research and School of Health Sciences, University of Notre Dame Australia, 19 Mouat Street, Fremantle, WA 6959, Australia.