A 6.4MB duplication of the alpha-synuclein locus causing fronto-temporal dementia and parkinsonism - phenotype-genotype correlations

Abstract

Importance

SNCA locus duplications are associated with variable clinical features and reduced penetrance but the reasons underlying this variability are unknown.

Objective

1) To report a novel family carrying a heterozygous 6.4Mb duplication of the SNCA locus with an atypical clinical presentation strongly reminiscent of frontotemporal dementia (FTD) and late-onset pallidopyramidal syndromes. 2) To study phenotype-genotype correlations in SNCA locus duplications.

Design, Setting, Participants and Data sources

We report the clinical and neuropathologic features of a family carrying a 6.4Mb duplication of the SNCA locus. To identify candidate disease modifiers, we undertake a genetic analysis in the family and conduct statistical analysis on previously published cases carrying SNCA locus duplication using regression modelling with robust standard errors to account for clustering at the family level.

Main outcome measures

To assess whether length of the SNCA locus duplication influences disease penetrance and severity, and whether extra-duplication factors have a disease-modifying role.

Results

We identified a large 6.4Mb duplication of the SNCA locus in this family. Neuropathological analysis showed extensive α-synuclein pathology with minimal phospho-tau pathology. Genetic analysis showed an increased burden of PD-related risk factors and the disease-predisposing H1/H1 MAPT haplotype. Statistical analysis of previously published cases suggested that there is a trend towards increasing disease severity and disease penetrance with increasing duplication size. The corresponding odds ratios (95% CI) from the univariate analyses were 1.17 (0.81 to 1.68) and 1.34 (0.78 to 2.31) respectively. Gender was significantly associated with both disease risk and severity; males compared to females had increased disease risk and severity and the corresponding odds ratios (95% CI) from the univariate analyses were 8.36 (1.97 to 35.42) and 5.55 (1.39 to 22.22) respectively.

Conclusions and relevance

These findings further expand the phenotypic spectrum of SNCA locus duplications. Increased dosage of genes located within the duplicated region probably cannot increase disease risk and disease severity without the contribution of additional risk factors. Identification of disease modifiers accounting for the substantial phenotypic heterogeneity of patients with SNCA locus duplications could provide insight into molecular events involved in α-synuclein aggregation.

INTRODUCTION

α-Synuclein is a protein central to the pathogenesis of Parkinson’s disease (PD), a role illustrated by the identification of α-Synuclein as the principal component of Lewy Bodies (LBs) ¹, the pathological hallmark of PD, and of α-Synuclein (SNCA) point mutations and multiplications as rare causes of PD ^2-4. Disease severity correlates with the number of SNCA alleles in multiplication carriers. Patients with SNCA triplication present with an early-onset, aggressive form of PD, with dementia, psychiatric features and dysautonomia ⁵. On the other hand, SNCA duplications are not fully penetrant and are associated with variable clinical features ranging from late-onset sporadic PD to presentations indistinguishable from those in triplication carriers but the reasons underlying this variability are unknown.

Here, we report a new kindred carrying the largest non-chromosomal duplication of the SNCA locus identified to date presenting with clinical features reminiscent of frontotemporal dementia (FTD) and widespread Lewy bodies. We undertake a comprehensive assessment of candidate disease modifiers in carriers of SNCA locus duplications based on genetic analysis of the proband and on statistical analysis of previously published cases.

METHODS

Genetics

MAPT was screened through Sanger sequencing in cases III:1 and III:3 (figure 1A) because of clinical similarity to FTD ⁶. Because of clinical similarities to SNCA multiplication cases and widespread α-synuclein pathology, a genomic DNA sample from case III:1 was assessed for whole gene SNCA multiplications with the Multiplex-ligation dependent probe amplification (MLPA) kit P051C (MRC Holland, the Netherlands) ⁷. The approximate breakpoints of the duplication were determined through an Agilent custom 8×60k dense comparative genomic hybridization (CGH) array designed as part of a larger on-going study with dense spacing of probes throughout the SNCA genomic region (CP, details available on request).

Figure 1. ‘Family tree and neuropathology findings’.

A) Family tree. Red arrow depicts the proband. Information has been omitted for deidentification purposes. B) Representative images of α-synuclein immunoreactive inclusions within (a) substantia nigra (SN), (b) locus coeulus (LC), (c) nucleus basalis of Meynert (NBM) and (d) transentorhinal cortex (tEC). Severe neuritic α-synuclein deposition is shown in the CA2 region of the hippocampus at low and high magnification (e, f). α–Synuclein coiled bodies shown in the white matter of the cerebellum (g,h, CBL). Sparse AT8 (phospho-tau, Ser 202 and Thr205) immunoreactive neurofibrillary tangles were observed in the transentorhinal cortex (i,j, tEC). Scale bars represent 50 μm. PD=Parkinson’s disease; FTD=Frontotemporal dementia

In an attempt to identify risk factors potentially influencing the clinical features, a DNA sample from case III:1 was genotyped on the Immunochip, an Illumina array of custom content with a specific focus on immune-related and PD/parkinsonism genes ^8-10. We focused on risk variants identified through previously published PD genome wide association studies (GWAS) and on mutations and risk factors located within PD genes (see supplementary information for methods).

Neuropathology

The brain from case III:3 was fixed by suspension in buffered 20% formalin and sampled extensively for neuropathology. Paraffin-embedded sections (8μm) from cortical, subcortical, brainstem and cerebellar regions were stained immunohistochemically for standard pathological markers including Aβ (DAKO, M0872) AT8 (Tau Ser202/Thr205, Source Bioscience 90206), TDP-43 (Abnova, H00023435-M01) and α-synuclein (Abcam ab15530). For Aβ and α-synuclein immunohistochemistry, sections required pre-treatment with formic acid for 10 minutes before antigen retrieval. Antigen retrieval was achieved by heating sections in a pressure cooker for 10 minutes in boiling citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with methanol/0.3% H₂O₂ followed by incubation in dried milk solution (10% in PBS, 30 minutes) to block non-specific antibody binding. Primary antibodies diluted in PBS were applied for 60 minutes at room temperature and staining was visualised by the streptavidin-biotin-peroxidase method (Vector laboratories, Peterborough, UK), using 3,3′-diaminobenzidine as the chromogen with Mayer’s haematoxylin counterstaining.

Statistical analysis of previously published cases

We reviewed previously published articles on carriers of SNCA locus duplications in order to assess whether the length of the duplication influences penetrance and disease severity and whether longer duplication sizes are related to a younger age at onset (AAO), indicating a contribution of increased dosage of other gene(s) to the development of disease. We used a duplication size threshold of 5Mb as an inclusion criterion which was chosen on the basis of the following arguments. Firstly, we hypothesised that shorter duplications are more frequently non-penetrant and that as duplication size increases, the individual exhibits a more severe phenotype ranging from unaffected, sporadic PD, PD-Dementia, to indistinguishable from triplication carriers. Secondly, we also hypothesised that, above a certain duplication length threshold, the individual will exhibit a more complex phenotype with features unusual for SNCA multiplications. Consequently, two individuals with such complex presentations, one described by Garraux et al (2012) ¹¹ and the case reported herein carrying duplications larger than 5Mb were excluded from this analysis. Published individuals were also excluded if any of the following were true: a) there was no information on the length of the duplication, b) there was insufficient clinical information, c) clinical presentation was clearly inconsistent with parkinsonism, d) they were affected relatives of probands with SNCA duplications but their carrier status wasn’t assessed (efigure 1). Data on all eligible individuals from each family carrying the duplications were included in the study to maximise study power (eTable 1). Review of the articles’ eligibility and cataloguing of the information was undertaken by the main author (EK) and was repeated twice.

Disease severity was measured by deriving a composite score for each affected individual from the published data. Each individual was assigned a score according to a clinical presentation point system as follows: Typical PD/parkinsonism = 1, young onset (before or at 40 years) = 1, dementia = 1, hallucinations = 1, dysautonomia = 1, depression = 1, any additional features (dystonia, epilepsy, sleep disturbances, myoclonus, psychiatric features) = 1. These scores were then summed up to form a composite score, which ranged from 0 (unaffected) to 6 (severely affected).

Numerical data were summarised using mean and standard deviation or median and range depending on data distribution. Categorical data were summarised using count and percentages. We used Spearman’s rank correlation to quantify strength of monotonic association between duplication size and each of AAO and disease severity. The main outcomes of interest were disease status (binary), disease severity (ordinal) and age at onset (binary). We modelled binary outcomes using logistic regression with robust sandwich estimation of the variance to account for the clustering effect at the family level ¹². We modelled ordinal outcomes using ordinal regression with the proportional odds assumption, adjusted for clusters at the family level ¹³. This approach has been shown to be more suitable than multilevel approaches in the presence of small number of clusters with low number of individuals ¹⁴. We investigated the individual effect of the covariates on each of the outcomes using univariate analyses. We also investigated their combined effect using multivariable analyses.

The functional form of the relationship between duplication size and each outcome was also evaluated graphically. Model fit was assessed using the Akaike Information Criterion (AIC) ¹⁵, where smaller AIC is preferred.

All statistical analyses were carried out using Stata ¹⁶. Results from modelling are presented as estimates (95 % confidence interval). Plots were generated with the statistical package R version 3.0.2 ¹⁷ using the package ggplot2 ¹⁸.

RESULTS

CASE REPORT

Clinical description

The proband, an English, Caucasian woman (patient III:3, figure 1A), first developed lifelong and progressive symptoms of extreme anxiety, panic disorder and hallucinations from age 8 that were managed adequately with medication up to her mid-20’s when she had to quit her job. At age 38 years, she developed right arm tremor followed by a progressive akinetic-rigid syndrome, plus a classic pill-rolling tremor, complicated by worsening obsessive behaviour, walking disturbances and falls, increased salivation, REM sleep behaviour disorder, personality changes, short-term memory impairment and poor self-care. She also had facial, head and tongue tremor accompanied by blepharospasm, dystonic neck and dysarthria. Levodopa treatment improved her walking but increased her falls. Neuropsychometry testing showed frontal and temporal deficits though neuroimaging was normal. There was a history of recurrent unexplained blackouts or seizures. EEG showed traces of alpha-rhythm, and autonomic testing indicated cardiovascular autonomic failure. On examination, she exhibited akinesia and rigidity of all four limbs, rest tremor and hypometric saccades. From the age of 42, the motor and cognitive aspects of her disorder declined further, with poor concentration, frontal release signs, wandering, repetitive speech and a profound increase in appetite particularly for sweet food, and at age 46 she was given a clinical diagnosis of possible FTDP-17 but no MAPT mutations were detected. She became bedbound and died at the age of 49 years.

In her family history, her father (II:3), paternal grandmother (I:1) and two paternal great-aunts (I:2, I:3) suffered from PD with no documented dementia. Her paternal cousin (III:1) was diagnosed as having possible FTDP-17 in his early 50’s when he presented with memory problems, hallucinations and falls, complicated by alcoholism. This progressed with increasing cognitive decline and disorientation and rigidity at which point his progressive condition was managed by local services.

Pathology

Neuropathological examination showed neuronal loss which was most severe in the substantia nigra, moderate in the locus coeruleus and dorsal motor nucleus of the vagus and mild in the nucleus basalis of Meynert and cerebellar Purkinje cells. Neuronal loss was not observed in other regions including the hippocampus. Widespread Lewy bodies and Lewy neurites affecting brainstem, limbic and neocortical regions corresponding to neocortical Lewy body pathology, Braak stage 6^19,20 (figure 1B, a-d) were detected. The hippocampus showed mild accumulation of α–synuclein inclusions with severe neuritic pathology in the CA2 region (figure 1B, e, f). Occasional oligodenrdrocytes contained α–synuclein immunoreactive inclusions with coiled body morphology. No inclusions resembling glial cytoplasmic inclusions were observed (figure 1B, g, h). Minimal phospho-tau pathology was present in the transentorhinal cortex corresponding to Braak and Braak stage I (figure 1B, I, j) and there were sparse diffuse cortical amyloid β deposits. TDP-43 pathology was not present.

Genetics

Patient III:1 was negative for MAPT point mutations but was found to carry a heterozygous SNCA duplication on MLPA (figure 2A). CGH arrays showed that the duplication extended over approximately 6.4Mb (chr4:88349207-94751141, corresponding to the first and the last duplicated probes respectively, human build 37) and contained 37 genes including SNCA (figure 2B,C)(table 1). The first and last non-duplicated probes are A_14_P119434 (chr4:88295927-88295986) and A_14_P124103 (chr4:94859877-94859936) respectively (figure 2D). The breakpoints are located centromeric to intron 1 of the NUDT9 gene (NM_198038.2), and telomeric to the ATOH1 gene (figure 2E).

Figure 2. ‘Genetic findings’.

A) MLPA results depicting the duplication. B) Minimum duplicated region as identified by the CGH array (chr4:88,349,207-94,751,141, HB37). C) Minimum duplicated region as identified by the Immunochip (chr4: 88,231,429-94,816,144 HB37) with an increased logR ratio and probes forming 4 distinct genotype clusters ⁴⁶. Region depicted in figure is chr4: 83,666,791-97,713,411 (HB37). D) Genes included within the minimum duplicated region (chr4:88,349,207-94,751,141, HB37) as identified by the CGH array. E) Breakpoint regions and repetitive sequences identified through UCSC repeat masker. Upper panel centromeric (chr4:88,249,207-88,349,207), lower panel telomeric (chr4: 94,751,141-94,851,141). F) Comparison of the length of the duplicated/triplicated region between our case and published cases (band sizes are approximate). Sizes represent minimum length based on Human Build 36. G) Location of breakpoints. Height of peak corresponds to the number of cases with a breakpoint in the particular region. Breakpoint location could correspond to regions of recombination hotspots ^21,47. The case reported by Garraux et al (2012) ¹¹ was excluded from both images.

Table 1.

List of genes included in the duplicated region in case III:1. Generated with NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/projects/mapview/).

List of genes included within duplication region in case III:1
Start1	Stop1	Gene Symbol	Description	Known role in disease
88343728	88380606	NUDT9	nudix (nucleoside diphosphate linked moiety X)-type motif 9	NA
88394487	88450655	SPARCL1	SPARC-like 1 (hevin)	NA
88529681	88538025	DSPP	dentin sialophosphoprotein	dentinogenesis imperfecta-148,49
88571454	88585513	DMP1	dentin matrix acidic phosphoprotein 1	Autosomal recessive hypophosphatemia50
88707075	88707514	CHCHD2P7	coiled-coil-helix-coiled-coil-helix domain containing 2 pseudogene 7	NA
88720702	88733601	IBSP	integrin-binding sialoprotein	NA
88742563	88767969	MEPE	matrix extracellular phosphoglycoprotein	NA
88812995	88815167	HSP90AB3P	heat shock protein 90kDa alpha (cytosolic), class B member 3, pseudogene	NA
88896802	88904563	SPP1	secreted phosphoprotein 1	NA
88928799	88998931	PKD2	polycystic kidney disease 2 (autosomal dominant)	Autosomal dominant polycystic kidney disease (ADPKD) 51
89011416	89152474	ABCG2	ATP-binding cassette, sub-family G (WHITE), member 2	NA
89084691	89085162	RPL31P24	ribosomal protein L31 pseudogene 24	NA
89178761	89205983	PPM1K	protein phosphatase, Mg2+/Mn2+ dependent, 1K	NA
89299891	89364249	HERC6	HECT and RLD domain containing E3 ubiquitin protein ligase family member 6	NA
89378268	89427321	HERC5	HECT and RLD domain containing E3 ubiquitin protein ligase 5	NA
89428154	89431638	LOC728333	nuclear receptor coactivator 4 pseudogene	NA
89442129	89444952	PIGY	phosphatidylinositol glycan anchor biosynthesis, class Y	NA
89442129	89444952	PYURF	PIGY upstream reading frame	NA
89448713	89449318	LOC100129137	CD53 molecule pseudogene	NA
89513647	89629686	HERC3	HECT and RLD domain containing E3 ubiquitin protein ligase 3	NA
89605999	89606105	RNU6-33P	RNA, U6 small nuclear 33, pseudogene	NA
89617066	89619023	NAP1L5	nucleosome assembly protein 1-like 5	NA
89630940	89651254	FAM13A-AS1	FAM13A antisense RNA 1	NA
89647105	89978346	FAM13A	family with sequence similarity 13, member A	NA
90031864	90033424	LOC731282	uncharacterized LOC731282	NA
90033968	90036052	TIGD2	tigger transposable element derived 2	NA
90165429	90229161	GPRIN3	GPRIN family member 3	NA
90645250	90759447	SNCA	synuclein, alpha	PD 2
90757552	90763142	LOC644248	uncharacterized LOC644248	NA
90816052	90875780	MMRN1	multimerin 1	NA
91048684	92523370	CCSER1	coiled-coil serine-rich protein 1	NA
91759636	91760266	TMSB4XP8	thymosin beta 4, X-linked pseudogene 8	NA
93103426	93105206	LOC133083	peptidase (mitochondrial processing) alpha pseudogene	NA
93225550	94693649	GRID2	glutamate receptor, ionotropic, delta 2	Cerebellar ataxia 52
93623496	93623786	MTND1P19	MT-ND1 pseudogene 19	NA
93743117	93744164	LOC100422562	guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 pseudogene	NA
94750078	94751142	ATOH1	atonal homolog 1 (Drosophila)	NA

¹Base pairs, HB37

The patient was homozygous for the wild type allele for all rare SNPs or probes encoding mutations on the Immunochip apart from 1 heterozygous parkin variant that is probably non-pathogenic (rs1801334) (MAF 0.04 in European populations as catalogued in ensembl) (eTable 2) and had a MAPT haplotype of H1/H1. The patient carried a total of 18/46 risk alleles for 23 GWAS SNPs (eTable 3) placing him in the 3rd risk quintile shown to confer approximately a 77% increase in PD risk^21-23.

Statistical analysis

Table 2 describes the demographic and clinical characteristics of individuals included in the statistical analysis. Overall, 73% (27/37) of individuals were affected, with a higher proportion of females compared to males in the unaffected group (90% versus 10 %). The median (range) age at onset of disease was 47 (31-71) years. The median (range) composite score was 2 (0-6). The median (range) duplication size for affected and unaffected individuals was 0.63 (0.2-5) and 0.6 (0.4-3.47) Mb respectively. There was a weak positive monotonic association between duplication size and composite score (rho=0.17, p=0.314) and between duplication size and age at onset (rho=0.12, p=0.560).

Table 2. Demographic and clinical characteristics of study sample by disease status.

Demographic and clinical characteristics of individuals included in the statistical analysis.

	Disease status		Overall
Variable	Unaffected	Affected
Individuals [n (%)]	10 (27%)	27 (73%)	37 (100%)
Gender
Male [n (%)]	1 (7.14%)	13 (92.86%)	14 (37.84%)
Female [n (%)]	9 (39.13%)	14 (60.87%)	23 (62.16%)
Members per family [median (range)]	NA	NA	2 (1-7)
Age at Onset [median (range)]	NA	47 (31-71)	47 (31-71)
Duplication size [median (range)]2	0.6 (0.4-3.47)	0.63 (0.2-5)	0.6 (0.2-5)
Composite score [median (range)]	0 (0-0)	2 (0-6)	2 (0-6)

²Mean (standard deviation-SD): 1.17 (1.22) (unaffected), 2.09 (2.12) (affected), 1.84 (1.95) (overall)

Table 3 provides estimates of the effect of duplication size and gender on disease status, age at onset and disease severity. Overall, only the effect of gender on disease status and disease severity reached statistical significance for both univariate and multivariate models (p<0.05). The odds of being affected were more than 8-fold for males compared to females. The corresponding OR (95% CI) was 8.36 (1.97 to 35.42). The odds of being affected increased by approximately 34% for each unit increase in duplication size. The corresponding OR (95% CI) was 1.34 (0.78 to 2.31). Males had a 77% greater chance of developing an early onset form of disease in comparison to females with an OR of 0.23 (95% CI: 0.04 to 1.28), whereas increase in duplication size by one unit resulted in a 6% decrease in the chance of developing late-onset disease (OR, 95% CI: 0.94, 0.71 to 1.24). Males had over 5-fold increase in the chance of developing a more severe disease, and disease severity increased by 17% for each unit increase in duplication size. The corresponding OR (95% CI) were 5.55 (1.39, 22.22) and 1.17 (0.81, 1.68) respectively. (All ORs quoted in-text refer to univariate models).

Table 3.

Estimates for univariate (A) and multivariate models (B). OR represents the odds of affected disease status or late age at onset (> 40 years) for models 1 and 2 respectively (logistic regression models); OR >1 represents the odds of being affected or having a late AAO. OR represents the odds of severe disease outcome compared to mild and moderate for model 3 (ordinal logistic regression), where OR > 1 indicates worse outcome. OR=odds ratio, 95% CI=95% confidence intervals, AIC= Akaike Information Criterion.

A. Estimates for Univariate models
	Model 1			Model 2			Model 3
Outcome	Disease status (Logistic regression with robust standard errors)			Age at Onset (Logistic regression with robust standard errors)			Disease severity (Ordinal logistic regression with robust standard errors)
Independent variable	OR (95% CI)	p-value	AIC	OR (95% CI)	p-value	AIC	OR (95% CI)	p-value	AIC
Duplication size	1.34 (0.78, 2.31)	0.285	45.31	0.94 (0.71, 1.24)	0.662	39.49	1.17 (0.81, 1.68)	0.395	83.78
Gender
female	base			base			base
male	8.36 (1.97, 35.42)	0.004**	41.99	0.23 (0.04, 1.28)	0.094	36.49	5.55 (1.39, 22.22)	0.016*	78.09
B. Estimates for Multivariate models
Duplication size	1.35 (0.77, 2.37)	0.297	42.40	0.95 (0.69, 1.31)	0.766	38.43	1.12 (0.78, 1.62)	0.532	79.61
Gender
female	base			base			base
male	8.26 (1.81, 37.75)	0.006**		0.24 (0.04, 1.29)	0.095		5.27 (1.29, 21.50)	0.021*

DISCUSSION AND CONCLUSION

A total of 29 kindreds carrying SNCA locus duplications have been reported in the literature ²⁴, with pathology information on 4 duplication patients (3 belonging to the same family) ^25-29 (eTable 4). The size of the duplicated region has been studied in 16 kindreds/sporadic cases (eTable 4) and has been found to vary greatly, from 0.2Mb ³⁰to 41.2Mb ¹¹ containing from 2 to 150 genes (figure 2E,F, eTable 5). The case described here carries the largest ‘submicroscopic’ duplication of the SNCA locus reported to date.

Even though we were not able to determine the exact breakpoints of the duplication, in accordance with Ibanez et al (2009) ³¹, we showed that these lay within regions rich in repetitive elements and especially transposable elements (long interspersed elements-LINEs, short interspersed elements-SINEs, and long terminal repeat-LTR retrotransposons) facilitating the insertion of additional copies of flanking sequences in the genome ³², allowing non-homologous recombination events leading to genomic structural rearrangements. The 4q22 region is known to be inherently prone to disruption as a fragile site (FRA4F), which interestingly was originally described as a 7 Mb region corresponding very closely to the duplicated region in this report ³³. Subsequent work has extended the fragile site from 88200000-99100000Mb ³⁴ but the centromeric boundary still corresponds to the centromeric breakpoint in our report. Other reported copy number variants (CNVs) are mostly within the confines of the fragile site, although a breakpoint centromeric to the proposed FRA4F boundary has been documented in some cases (figure 2F, G).

This report further emphasises the clinical variability and expands the phenotypic spectrum associated with SNCA duplications and represents the first case with such a complex presentation and substantial overlap with other dementing and psychiatric disorders. SNCA duplications are characterised by incomplete penetrance and variable clinical presentation ^27,35 occasionally being indistinguishable from sporadic PD ³¹ or having the core clinical features of young-onset PD-dementia (YOPD-dementia) with dysautonomia, depression and hallucinations, resembling the phenotype of SNCA triplications ⁵. Our case exhibits some of the main features associated with SNCA multiplications (YOPD-dementia, hallucinations, dysautonomia) which are, however, part of a more complicated phenotype reminiscent of FTD. Obsessive compulsive disorder (OCD) and anxiety have not been previously reported in any patient with an SNCA duplication, nor was it present in any other member of our family. Thus although it is possible these were causally related in our case III:3, it is more likely a coincidental occurrence. However, this does make it difficult to establish with certainty the exact age at onset of the patient (possibly age 8 or probably age 38 years). Intrafamilial variability is marked as two members had an FTD-like phenotype and 4 family members from older generations apparently had typical PD. CaseII:1, an obligate carrier, was an alcoholic, as was his son III:1, and died at age 70 without documented parkinsonism or dementia.

Neuropathological examination showed features corresponding to idiopathic PD and SNCA duplication cases, with widespread Lewy pathology extending into neocortical regions (Braak stage 6), and no TDP-43 pathology ^25,27-29. There was only minimal Alzheimer pathology with tau pathology limited to Braak and Braak stage I and sparse diffuse amyloid β deposition in the cortex. Although previous studies have suggested ‘cross-seeding’ between α-synuclein and tau ³⁶, the relatively low accumulation of tau compared to α-synuclein containing inclusions suggests that this tau accumulation was a normal consequence of aging. Unlike other reported cases of SNCA duplication, neuronal loss was restricted and did not affect the hippocampus ^25,28. In further contrast, oligodendroglial inclusions with coiled body morphology were observed, but there were no structures resembling glial cytoplasmic inclusions²⁵. However, as has been reported, hippocampal pathology, particularly neuritic α–synuclein deposition was severe, especially in the CA2 region.

Given that SNCA duplications are frequently non-penetrant ^24,27 it is possible that mutations in another gene are responsible for the unusual clinical presentation of the proband. However, there are several lines of evidence supporting the pathogenicity of the duplication: the widespread Lewy body pathology and other pathological features are consistent with SNCA duplications, the pattern of inheritance of the disease in the family is autosomal dominant with possible reduced penetrance, and other affected relatives have phenotypes that have been previously associated with SNCA duplication. Thus, even though we were unable to confirm complete segregation due to the lack of DNA samples, the SNCA duplication is most likely to be responsible for the disease in this family.

The reasons underlying this clinical variability associated with SNCA duplication are currently unknown, although several preliminary hypotheses emerge from our analyses that require validation in future studies on larger cohorts of SNCA duplication carriers. We found trends for association between duplication size and disease status, age at onset and disease severity that didn’t reach statistical significance though association with gender did. Even though our study was underpowered to detect association (n=37) ^37,38, it is possible that larger duplication size may not independently produce disease without the contribution of additional risk factors. Gender-specific risk factors are an intriguing possibility as regression model fitting was improved after adjusting for gender (table 3), and males with SNCA duplications had an 8-fold increase in risk for developing PD. A predilection for male involvement is well known for sporadic PD ^39,40. Alternatively, an increased burden of other risk factors could have a disease-modifying role; the patient we report carried one single heterozygous parkin rare variant, had an H1H1 MAPT haplotype and an accumulation of risk alleles in loci identified through GWAS. Similarly, Itokawa et al have reported a patient with a SNCA duplication carrying a PD-risk factor (p.G2385R) within LRRK2 ³⁵. Other unexplored possibilities include increased dosage of common or rare variants with a regulatory effect on expression or other properties of α-synuclein ^41,42, mosaicism ⁴³, environmental factors ⁴⁴, or stochasticity ⁴⁵.

In conclusion, patients in whom complex presentations of onset in early adulthood with dementia and parkinsonism complicated by multiple cognitive, psychiatric and motor features should be considered for genetic testing for SNCA multiplication. Understanding of the genetic modifiers influencing the phenotype of SNCA duplications may be important in elucidating the mechanisms underlying α-synuclein accumulation and the formation of Lewy bodies.

Footnotes

Conflict of interest: Kailash P Bhatia received funding for travel from GlaxoSmithKline, Orion Corporation, Ipsen, and Merz Pharmaceuticals, LLC; serves on the editorial boards of Movement Disorders and Therapeutic Advances in Neurological Disorders; receives royalties from the publication of Oxford Specialist Handbook of Parkinson’s Disease and Other Movement Disorders (Oxford University Press, 2008); received speaker honoraria from GlaxoSmithKline, Ipsen, Merz Pharmaceuticals, LLC, and Sun Pharmaceutical Industries Ltd.; personal compensation for scientific advisory board for GSK and Boehringer Ingelheim; received research support from Ipsen and from the Halley Stewart Trust through Dystonia Society UK, and the Wellcome Trust MRC strategic neurodegenerative disease initiative award (Ref. number WT089698), a grant from the Dystonia Coalition and a grant from Parkinson’s UK (Ref. number G-1009).

URLs 

UCSC genome browser http://genome.ucsc.edu/

R http://www.r-project.org/

R package ggplot2 http://cran.r-project.org/web/packages/ggplot2/index.html

STATA package gologit2 http://www3.nd.edu/~rwilliam/gologit2/