TOMM40‘523’ poly-T repeat length is a determinant of longitudinal cognitive decline in Parkinson’s disease

The translocase of outer mitochondrial membrane 40 (TOMM40) ‘523’ polymorphism has previously been associated with age of Alzheimer’s disease onset and cognitive functioning in non-pathological ageing, but has not been explored as a candidate risk marker for cognitive decline in Parkinson’s disease (PD). Therefore, this longitudinal study investigated the role of the ‘523’ variant in cognitive decline in a patient cohort from the Parkinson’s Progression Markers Initiative. As such, a group of 368 people with PD were assessed annually for cognitive performance using multiple neuropsychological protocols, and were genotyped for the TOMM40 ‘523’ variant using whole-genome sequencing data. Covariate-adjusted generalised linear mixed models were utilised to examine the relationship between TOMM40 ‘523’ allele lengths and cognitive scores, while taking into account the APOE ε genotype. Cognitive scores declined over the 5-year study period and were lower in males than in females. When accounting for APOE ε4, the TOMM40 ‘523’ variant was not robustly associated with overall cognitive performance. However, in APOE ε3/ε3 carriers, who accounted for ~60% of the whole cohort, carriage of shorter ‘523’ alleles was associated with more severe cognitive decline in both sexes, while carriage of the longer alleles in females were associated with better preservation of global cognition and a number of cognitive sub-domains, and with a delay in progression to dementia. The findings indicate that when taken in conjunction with the APOE genotype, TOMM40 ‘523’ allele length is a significant independent determinant and marker for the trajectory of cognitive decline and risk of dementia in PD.


INTRODUCTION
Cognitive impairment is a notable symptom of Parkinson's disease (PD), not only because of its impact on patient-perceived quality of life, but also because significant dysfunction will affect a high proportion of people with PD (PwP) as the disease progresses 1,2 . Past research has reported a marked degree of clinical heterogeneity and different patterns of brain pathology have been identified as the underlying basis for cognitive decline in PD [3][4][5] , suggestive of a variable underlying pathophysiology and a complex interaction between different risk factors, including genetic variability. Candidate genes may account for a portion of susceptibility to cognitive impairment in PwP, risk of Parkinson's disease dementia (PDD), and for the pattern of decline within certain cognitive domains and rate of decline [6][7][8] .
Prior candidate genetic studies and genome-wide association studies have been central in identifying genetic factors that contribute to the development of cognitive dysfunction in PD, in particular the significant role of Apolipoprotein E Epsilon 4 (APOE ε4) in modulating risk of cognitive decline [9][10][11] . However, alternative approaches, including analysis of structural variants 12 and variation within noncoding regions of the genome 13,14 may also help to elucidate complex phenotypic heterogeneity 15 . One structural variant of note is the 'translocase of outer mitochondrial membrane 40' (TOMM40) polymorphic T-tract length (rs10524523, '523'); an intronic variant known to improve precision when estimating the age of onset of Alzheimer's disease (AD) in APOE ε3 carriers and to be associated with changes in cognition in nonpathological ageing [16][17][18][19][20][21][22] . While the functional effects of polymorphism at the '523' locus remain largely unknown, a reduction in the TOMM40 protein due to the decreased TOMM40 expression is known to be associated with α-synuclein accumulation and a number of other effects, including increased reactive oxygen species formation, oxidative damage, reduced mitochondrial integrity and neuroinflammation [23][24][25][26][27] . In view of the purported functional impact of allelic variation at the '523' locus, coupled with the shared clinical, pathological and molecular features of AD and PD [28][29][30] , polymorphism at this site is therefore a plausible candidate to modulate the development and progression of cognitive impairment in PD. Though, it is important to note that consideration of the effect of APOE must be taken into account, as previously reported 16 .
Our group, along with others, have previously demonstrated that polymorphism at the TOMM40 '523' locus is not a determinant of PD risk, although it may be a modifier of the age of symptom onset [31][32][33] . However, studies examining the possible role of '523' alleles in modulating cognitive decline in PD are fundamentally lacking, with only a single cross-sectional study reporting a lack of association with dementia in a mixed PD and diffuse Lewy body disease (DLB) cohort 34 . Furthermore, with sexual dimorphism being an increasingly reported phenomenon in the context of both cognition [35][36][37] , as well as in the cognitive status of people with PD [38][39][40] , it is imperative to consider such factors. As such, the primary objective of the present study was to examine the role of TOMM40 '523' alleles as determinants of the severity and trajectory of cognitive decline in a large, well-documented Caucasian PD cohort, and to determine whether the alleles are associated with differential effects on specific cognitive domains, when taking patient sex into account. Baseline and longitudinal clinical and demographic information  and cognitive scores for the 368 participants are presented in  Supplementary Tables 2 and 3. At baseline, the majority of participants were males (65.5%), had an average age of PD symptom onset of 59 Subsequently, in adjusted generalised linear mixed models (GLMMs), carriage of S alleles was associated with a significantly greater decline in the MoCA score over the study period (p = 0.009; Bonferroni corrected p = 0.036), and decline in HVLT (p = 0.017), and SFCOM scores (p = 0.001; Bonferroni corrected p = 0.004; Table 1), though the decline in HVLT did not remain statistically significant after Bonferroni correction. Conversely, carriage of L alleles was associated with superior scoring in the BJLO and LNS tests over the study period (p < 0.001 and p < 0.001, respectively; Bonferroni corrected p = 0.004 and p = 0.004, respectively). In unadjusted linear models, S and VL alleles were associated with better performance in the LNS (p = 0.015) and SDMT tests (p = 0.012), respectively, but these associations were not confirmed after adjusted for covariates and multiple comparison correction (Table 1).

Baseline demographic and cognitive performance data
TOMM40 '523' alleles and cognitive performance in APOE ε3/ ε3 carriers As previous literature has indicated that the predictive value of TOMM40 '523' in late-onset AD primarily applies to carriers of APOE ε3, analyses were conducted again within the dominant subgroup of APOE ε3/ε3 carriers (n = 205). In this group, unadjusted linear models also demonstrated that presence of the S alleles was associated with more severe global cognitive decline (p < 0.001; Bonferroni corrected p = 0.004), as measured by the MoCA, and with more severe decline in HVLT (p = 0.044), SFCOM (p = 0.009; Bonferroni corrected p = 0.036) and SDMT (p = 0.001; Bonferroni corrected p = 0.004) scores over time (Table 2), though decline in HLVT did not remain statistically significant after Bonferroni correction. These associations were confirmed in covariateadjusted models: MoCA (p < 0.001), SFCOM (p = 0.03) and SDMT (p = 0.022; Table 2).
In contrast, carriage of longer '523' alleles were associated with preservation of cognitive performance in a number of domains.
Unadjusted modelling disclosed a positive association between VL alleles and HVLT (p < 0.001), LNS (p = 0.006) and SDMT (p < 0.001) scores over time (Table 2). After allowing for covariates, only the association between VL alleles and better performance in the SDMT test remained significant (p = 0.005; Bonferroni corrected p = 0.020).
Sex-specific effects of TOMM40 '523' alleles on longitudinal cognitive performance in APOE ε3/ε3 carriers As previous literature has indicated that cognitive ability and cognitive decline significantly differ between males and females with PD 40 , analyses were conducted again considering the sexes as separate groups. The results are summarised in Table 3 and in  Supplementary Tables 5 and 6. While in males, unadjusted GLMMs revealed a number of significant associations between '523' alleles and cognitive function, only the association between carriage of S alleles and more severe decline in the MoCA remained significant after adjusted for covariates (p = 0.011; Table 3).
Notably, in females a number of associations were apparent between carriage of longer '523' alleles and cognitive ability over time. Unadjusted models revealed significant associations between L alleles and worse decline in the HVLT (p = 0.033) assessment (Supplementary Table 6), whereas carriage of VL alleles was associated with significantly better performance over time with the MoCA (p = 0.024), the BJLO (p = 0.039), SFCOM (p = 0.004) and LNS (p = 0.045) assessments (Supplementary Table 6). Multivariable GLMMs confirmed that carriage of S alleles was associated with significantly poorer performance in the MoCA over time (p = 0.006; Table 3; Bonferroni corrected p = 0.024). Finally, carriers of VL alleles had significantly better longitudinal cognitive performance with the MoCA (p = 0.002; Bonferroni corrected p = 0.008), BJLO (p = 0.050), SFCOM (p = 0.001; Bonferroni corrected p = 0.004), LNS (p = 0.024) and SDMT (p = 0.020) assessments, though BJLO, LNS and SDMT did not remain significant after correction for multiple comparisons.
Effects of TOMM40 '523' alleles on progression to PDD The full cohort was subsequently analysed to examine the effect of '523' alleles on progression to PDD. As most baseline cognitive scores were found to differ significantly between males and females (Supplementary Table 2), analyses were carried out with and without stratification by sex.
Firstly, combined '523' allele lengths were compared between the group with PDD and the group without dementia (PD-ND; Supplementary Table 7). When gender was combined, no significant differences were observable between the PDD and PD-ND groups (50.88 ± 14.01 and 52.01 ± 13.62, respectively). When separated by sex, combined '523' allele length did not significantly differ in males with PDD vs PD-ND (51.66 ± 14.14 and 51.67 ± 13.82, respectively), whereas in females there was a significant difference in combined allele length between females with PDD (48.03 ± 13.29) and females with PD-ND (52.59 ± 13.22; p = 0.004; Bonferroni corrected p = 0.016), pointing to a protective effect of longer alleles in females only.
In subsequent survival analyses, taking PDD diagnosis as the endpoint, no significant differences were exhibited between '523' S, L or VL carrier status when the sex of the Parkinson's Progression Markers Initiative (PPMI) cohort was combined (p = 0.944, p = 0.271 and p = 0.549, respectively). Analyses were re-run using Cox proportional hazard regression models, adjusting for APOE ε4 carrier status, and no significant associations were noted (S allele, p = 0.805; L allele, p = 0.192; VL allele, p = 0.759). After separating by sex, no significant associations were observable between '523' allele carrier status in males, using the Kaplan-Meier method ( which adjusted for APOE ε4 carrier status. In females, there were no significant observable effects with carriage of the S or L alleles using the Kaplan-Meier method, though there was a trend towards more rapid progression to dementia in females with the S allele ( Fig. 1D, S allele, p = 0.135; Fig. 1E, L allele, p = 0.709). Cox proportional hazard regression found no significant effect when adjusting for APOE ε4 carrier status (S allele, p = 0.218; L allele, p = 0.924). Notably, a significantly faster progression to dementia was apparent in females who did not carry VL alleles (Fig. 1F, VL allele, p = 0.033). This association remained significant when using Cox proportional hazard regression (p = 0.013) and after adjusting for carriage of APOE ε4, confirming that VL alleles may be protective. Following Bonferroni correction these associations were indicative of a trend, though not statistically significant.
TOMM40 '523' alleles and progression to PDD in APOE ε3/ε3 carriers Based upon PDD diagnosis, combined '523' allele lengths were compared between PDD vs PD-ND groups. When gender was combined, a significant difference was observed between PDD and PD-ND (49.32 ± 14.42 and 51.33 ± 14.52, respectively; p = 0.036). When separated by sex, males with PDD and PD-ND exhibited no significant differences in combined '523' allele length (49.28 ± 14.62 and 50.20 ± 14.95, respectively; p = 0.388). However, a significant difference in combined allele length was seen in females with PDD (49.42 ± 13.99) compared to females with PD-ND (53.25 ± 13.57; p = 0.043). While this association did not remain significant after Bonferroni correction (p = 0.172), it is suggestive of a protective effect of longer alleles.
Survival analysis using the Kaplan-Meier method, taking PDD diagnosis as the endpoints, showed no significant differences between S, L or VL carrier status when the sex of the PPMI cohort was combined (p = 0.396, p = 0.211 and p = 0.217, respectively). Cox proportional hazard regression models adjusting for sex also failed to show any significant associations (S allele, p = 0.485; L allele, p = 0.097; VL allele, p = .390). When considering males and females separately, and after excluding carriers of the L allele due to low numbers, no significant associations were observable between '523' allele carrier status and progression to PDD in males (S allele, p = 0.587; VL allele, p = 0.720). Though no significance was seen in female carriage of the S allele (S allele, p = 0.618), a trend towards more rapid progression was observed in females lacking the VL allele (VL allele, p = 0.152).

DISCUSSION
Cognitive impairment and progression to PDD are significant determinants of morbidity and quality of life in PD. The genetic contribution to these non-motor aspects of PD is an area of great importance. Within studies examining cognitive decline in nonpathological ageing populations [19][20][21][22] and in cohorts of individuals with AD 17,18 , the mitochondrial variant TOMM40 '523' has been implicated with mixed results. By comparison, two studies have found no association between the TOMM40 '523' and PD risk 32,33 , while a third group found that the L/VL '523' genotype was overrepresented in Swedish PD patients when compared to controls 31 . However, research into whether TOMM40 '523' alleles play a role in modulating cognitive impairment and rate of decline within PD is profoundly scant, with only a single cross-sectional study of a mixed PD and DLB cohort reporting that TOMM40 '523' is not associated with risk of dementia 34 . To our knowledge, no previous studies have explored the relationship between TOMM40 '523' poly-T length variability and domain-specific cognitive decline or progression to dementia in PD, or differential effects of TOMM40 '523' alleles on cognition in males and females. As such, this is the first study to assess this relationship in a large, welldocumented PD cohort undergoing serial cognitive assessments over a 5-year period. Our findings indicate that the TOMM40 '523' polymorphism does play a part in modulating cognitive decline, and suggest that alleles of '523' have differential sex effects, with shorter alleles being associated with greater global cognitive decline in both males and females, whereas longer alleles have both global and domain-specific protective effects in females with PD.
In past literature, case-control analysis has found that while TOMM40 '523' does not modulate or influence the risk of developing PD, it may affect the age at which symptoms develop 32,33 . The findings of the present study provide evidence that polymorphism in TOMM40 '523' may also have significant effects on cognitive decline and progression to PDD, independent of the influence of APOE ε4. While our findings demonstrate an effect of '523' on cognitive measures over time, significant differences were seen in the trajectory of cognitive decline and progression to PDD in females. Specifically, survival analysis showed a trend towards an association of S allele carriage with faster progression to PDD, whereas carriage of VL alleles was significantly protective in terms of progression to PDD in females, but not males. Furthermore, combined '523' allele length was significantly shorter in females with PDD compared to those without dementia (PD-ND group).
In view of the linkage disequilibrium which exists between TOMM40 and APOE, in order to accurately assess the independent contribution of '523' allelic variation, APOE genotypes must be taken into account 16 . Thus, further analysis examined the subcohort of carriers of APOE ε3/ε3, which is the most common genotype and accounted for~60% of the present PD cohort. The aforementioned associations in the whole cohort were reflected in the assessment-specific analysis of the sub-cohort of APOE ε3/ε3 carriers, when adjusting for covariates. For instance, S allele carriage was associated with greater decline in global cognitive ability (MoCA), as well as language fluency (measured by SFCOM assessment) and processing speed and attention (SDMT); while possession of VL alleles was protective of processing speed and attention. However, it is likely that these findings were also being driven by gender, as a number of significant associations between the '523' variant and cognitive decline in females were not apparent in males. In particular, within the female APOE ε3/ε3 group, '523' S allele carriage was associated with a significantly worse overall MoCA score, whereas VL alleles were significantly protective in terms of overall cognition (MoCA score), as well as visuospatial function, language fluency, and superior executive function and working memory. Moreover, in the APOE ε3/ε3 subcohort, no '523' allele stratification was observable in males with PD, whereas VL alleles appeared to be associated with slower progression to PDD (though not statistically significant) in females. It is possible that with a larger sample size of APOE ε3/ε3 genotype carriers, this allele and sex association may be confirmed. Taken together, these findings suggest that increasing TOMM40 '523' allele length has a protective effect in terms of cognitive decline in people with PD, and this effect is more noticeable in females.
Interestingly, a shorter TOMM40 allele length appeared to be a risk factor for deterioration in cognitive performance over time in this PPMI cohort. Such findings appear to be reflective of those found in a smaller, cross-sectional study by Lindqvist and colleagues 34 , though this group reported that both S and VL alleles exhibited a trend towards being associated with dementia in a mixed PDD and DLB cohort. Overall, such variable associations between TOMM40 '523' and aspects of cognition must be interpreted with caution. Furthermore, it is important to consider underlying differences between the two cohorts that might explain variation in findings, such as the age at disease onset, as a number of studies in the literature have suggested that an older age of onset is more likely to be associated with cognitive impairment [41][42][43][44][45][46] . Although information on age of onset was not provided in the Lindqvist paper 34 , their patient cohort appears to have been older than the PPMI cohort, which was comprised of recently diagnosed PwP with a mean age of onset of 59.9 years.
The strengths of the present study are that it examined this relationship longitudinally in a large, homogeneous PD cohort, while considering the effect of the APOE ε4 carrier status, as well as the known variability of cognitive trajectory between males and females 40 . As such, the findings here support the notion that the TOMM40 '523' poly-T repeat variants do play a role in modulating cognitive decline in PwP, independently of the effects of APOE ε4.
As indicated by genetic studies, the role of mitochondrial dysfunction in PD aetiology and pathogenesis is increasingly wellrecognised. A recent study consistently observed that TOMM40 protein deficits in brain samples from PwP correlated with a higher number of mitochondrial DNA deletions, and with enhanced oxidative stress, reduced ATP production and abnormal complex I protein concentrations 25 . Furthermore, it has been shown that TOMM40-facilitated importation of α-synuclein into mitochondria inhibits mitochondrial complex I 26 , a common source of mitochondrial dysfunction in PD. In addition to this, TOMM40 is closely associated with the functioning of two familial PD genes, Parkin and PINK1, where TOMM40 is required by the PINK1 protein for mitochondrial localisation and Parkin recruitment in mitophagy 47 . Clearly, this variant may play a pathophysiological role in the disease risk and progression of PD. This, when combined with findings from studies that implicate the TOMM40 variant in AD, cognitive decline in the healthy ageing and in DLB, is why such a variant is proposed as a region of interest in the context of PD-related cognitive ability.
Certain limitations of the present study should be noted. Firstly, as the PPMI cohort comprised only recently diagnosed PwP (with a mean disease duration of~7 years and a mean age at onset of 60 years), the present findings need to be confirmed in broader, more representative community-based PD cohorts. Moreover, as the cohort we examined was purely Caucasian, further studies are required to determine whether the reported allelic associations are also present in other racial and ethnic groups. In addition, given it has been suggested that using WGS for genotyping TOMM40 '523' may be less accurate than PCR-based techniques for longer alleles 48 , further studies should compare allelic associations using the two methods. Though the study was based on the use of well-recognised and validated cognitive instruments, future studies may benefit from utilising more comprehensive cognitive assessment protocols or modern imaging technology. Finally, despite being a comprehensive cohort, analyses of the L allele presented with power issues and, in some analyses, had to be excluded. Thus, future analyses using even larger comprehensive cohorts are warranted.
Although the role of the TOMM40 '523' poly-T repeat has been well-explored in AD and healthy age-related cognitive decline, there is a paucity of studies investigating whether it has a role in cognitive decline in PD. The findings of the present longitudinal study in a large PD cohort from the PPMI provide the first evidence that the poly-T repeat length is a significant determinant of cognitive decline, independent of APOE4, and that the effects are sex-dependent. Thus, while short TOMM40 '523' alleles were associated with more severe cognitive decline in both sexes, in females, longer alleles were associated with protective effects on global cognition, as well as particular cognitive sub-domains, including attention and processing speed, language fluency, working memory. and executive and visuospatial functions, and appeared to delay progression to PDD. Our findings highlight the importance of the TOMM40 '523' polymorphism in cognitive decline in PD and suggest that further studies in other PD populations would be worthwhile. When combined with sex and APOE genotype, TOMM40 '523' may be a viable prognostic marker, and may assist in understanding the pathology underlying cognitive decline in PD.  TOMM40 rs10524523 and APOE ε genotyping

Statistical methods
The PPMI cohort was analysed using IBM-SPSS software (version 26, IBM Corporation). A significant nominal p value < 0.05 was employed for all statistical tests. Variables were described using mean and standard deviation (SD), or frequency and percent (%), as appropriate. Normality was assessed using the Shapiro-Wilk test, with subsequent clinical characteristics analysed using independent samples T test, Mann-Whitney U, Kruskal-Wallis or Chi-square, as appropriate.
Within longitudinal studies, trends of mean clinical assessments and patient clinical characteristics were assessed over time using GLMMs. Unadjusted GLMMs were used as univariate models to assess if clinical characteristics were significantly associated with cognitive examination scores over time. Variables identified as being statistically significant in univariate models, as well as APOE ε4 status, were considered covariates and were included in multivariable adjusted GLMMs. Variables included in multivariable models as covariates included years between assessments, age at assessment, age at disease onset, disease duration, gender, years of education and APOE ε4 status (0, 1 or 2 copies). For determining the presence of multicollinearity, the variance inflation factor (VIF) was calculated for all independent variables. For all reported models, VIF values were <2. Adjusted GLMMs were constructed to assess the impact of '523' genotype on cognition over time, independently of other covariates. As multiple comparisons were made in this exploratory study, we report both unadjusted p values, covariate-adjusted p values and covariate adjusted with Bonferroni corrected p values for each analysis. Bonferroni corrected p values were calculated by multiplying the p value to reflect the level of multiple hypotheses tested in this manuscript, being four levels. Furthermore, Akaike information criterion was used to compare model fit, where all multivariable models had lower values than unadjusted models, indicating better model fit. Residual plots were examined for all models and no violations were noted.
To evaluate the association between TOMM40 '523' genotype and progression to PDD, participants were stratified into PDD (dementia) or PD-ND (no dementia) groups, and mean combined '523' allele lengths were compared between these groups and survival curves were estimated by the Kaplan-Meier method. To compare the survival curves, the log-rank test was applied, placing weight on longer survival periods 64,65 . In addition, all distributions of ages at PDD onset were compared via Cox proportional hazard regression models, while adjusting for APOE ε4 status. All aforementioned statistical analyses were also carried out when separating the cohort by sex, which is previously reported to be a significant factor in cognitive impairment within PD 40 and in the sub-group of APOE ε3/ε3 carriers 16 .