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A new alpha-synuclein missense variant (Thr72Met) in two Turkish families with Parkinson’s disease

Introduction: Missense variants and multiplications of the alpha-synuclein gene (SNCA) are established as rare causes of autosomal dominant forms of Parkinson’s Disease (PD). Methods: Two families of Turkish origins with PD were studied; the SNCA coding region was analyzed by Sanger sequencing, and by whole exome sequencing (WES) in the index patient of the first and the second family, respectively. Co-segregation studies and haplotype analysis across the SNCA locus were carried out. Functional studies included in vitro thioflavin-T aggregation assay and in silico structural modelling of the alpha-synuclein (α-syn) protein. Results: We identified a novel heterozygous SNCA variant, c.215C > T (p.Thr72Met), segregating with PD in a total of four members in the two families. A shared haplotype across the SNCA locus was found among variant carriers, suggestive of a common ancestor. We next showed that the Thr72Met α-syn displays enhanced aggregation in-vitro, compared to the wild-type species. In silico analysis of a tetrameric α-syn structural model revealed that Threonine 72 lies in the tetrameric interface, and substitution with the much larger methionine residue could potentially destabilize the tetramer. Conclusion: We present clinical, genetic, and functional data supporting a causative role of the SNCA c.215C > T (p.Thr72Met) variant in familial PD. Testing for this variant in patients with PD, especially of Turkish origin, might detect additional carriers. Further functional analyses might offer new insights into the shared biochemical properties of the PD-causing SNCA missense variants, and how they lead to neurodegeneration.


Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by loss of neurons in the substantia nigra and other brain areas, and accumulation of intracellular inclusions containing misfolded alpha-synuclein (α-syn) protein, termed Lewy bodies and Lewy neurites [1]. The etiological landscape of PD is complex and incompletely known; however, the identification of rare disease-causing genes has contributed substantially to illuminate the underlying disease mechanisms and pathways [2].
Rare, highly penetrant variants in the gene encoding α-syn (SNCA) were the first identified cause of dominantly inherited PD. Since the first reported and most intensively studied missense substitution (p. Ala53Thr) in the "Contursi kindred" and additional Greek families [3], additional missense SNCA variants have been identified in patients with PD or related neurodegenerative disorders from several populations (Table 1). However, a disease-causing role has not been established for several of these variants, particularly those identified in single patients and without evidence of intra-familial co-segregation with disease. Genomic multiplications (duplications and more rarely triplications) encompassing the whole SNCA locus are a more frequent cause of PD than are the missense variants [4]. Last, common non-coding SNCA variants are one of the most relevant risk factors for the sporadic forms of The phenotypic spectrum associated with the rare, disease-causing SNCA variants is broad, encompassing PD (with or without atypical clinical signs), PD-dementia, dementia with Lewy bodies, frontotemporal dementia, and, more rarely, multiple system atrophy [6][7][8][9]. The mechanisms by which SNCA variants lead to neurodegeneration remain incompletely understood. Therefore, identification of additional PD-causing variants in this gene might provide new and important clues. Here, we report clinical, genetic, and protein expression data of a novel rare SNCA variant: c.215C > T (p.Thr72Met), identified in two families of Turkish origin with dominantly transmitted, late-onset PD and concomitant cognitive decline.

Materials and methods
Two unrelated families of Turkish origins with multiple members affected by PD and compatible with dominant inheritance were identified and clinically characterized, one (Family 1) at the Istanbul Faculty of Medicine, Turkey, and the other (Family 2) at the Erasmus MC, Rotterdam, The Netherlands.
Genomic DNA from 292 unrelated Turkish individuals free from PD was used to test for the frequency of the SNCA variant identified in the two PD families. Some of these samples (n = 56) were also used for haplotype analyses. These study procedures were approved by the relevant ethical authorities, and informed consent was obtained from all participating subjects.

Genetic studies
Genomic DNA was extracted from peripheral blood using standard protocols. In Family 1 (F1), Sanger sequencing of the coding and the flanking intronic regions of SNCA, LRRK2, and GBA was performed (PCR protocols and primers for SNCA Sanger sequencing are listed in Supplementary Table 1). The PCR products were then loaded on an ABI 3730XL Genetic Analyzer (Thermo Fisher Scientific). Sequences were analyzed using the software packages Seqscape v3⋅0 (Thermo Fisher Scientific) and Sequencing Analysis v6⋅0 (Thermo Fisher Scientific). SNCA variants were annotated according to the Gene Bank transcript accession number NM_000345 (NACP140). Variant annotation with Varcards was used for the in silico pathogenicity predictions [10]. The presence of copy number variations in known PD-causing genes was tested by Multiplex Ligation-dependent Probe Amplification (MLPA) technique, according to the MLPA General Protocol of MRC-Holland (https:// www.mrcholland.com/). We used the MRC Holland kits P051 and P052B that contain probes targeting multiple PD-causing genes. In Family 2 (F2), subject III-1 underwent testing by Whole Exome Sequencing (WES), filtered for variants in known PD-causing or PD-related genes (the list of genes tested can be found on Supplementary Table 2). In addition, one of the offspring of subject II-1 underwent NGS-based gene panel testing for inherited neuropathies.
For haplotype analysis, six short tandem repeat (STR) markers distributed in a region of~11 Mb and containing the SNCA gene were selected. The markers were amplified by PCR as described elsewhere [11]. PCR products were mixed with the GeneScan 500-LIZ Size Standard (Applied Biosystems), separated on an ABI 3730xl capillary sequencer (Applied Fevga et al. Page 3 Biosystems), and analyzed with GeneMarker (v2.4.0) (Softgenetics LLC, State College, PA, USA). PCR primers are provided in Supplementary Table 3. Four markers, tagging a haplotype shared between F1 and F2, were also typed in 56 unrelated Turkish controls to estimate the frequency of the shared haplotype in the Turkish population.

Alpha-synuclein (α-syn) protein studies
The expression and purification of GST-fused α-syn carrying the Thr72Met variant (α-syn Thr72Met) were carried out as previously described for the wild-type α-syn [12]. The p. Thr72Met substitution was created by site-directed mutagenesis using PCR and confirmed by DNA sequencing. Briefly, α-syn-Thr72Met was expressed in Rosetta 2 (DE3) E. coli (Novagen) by inducing with 1 mM iso-propyl-β-D-thiogalactopyranoside for 16 h at 20 °C. Cells were harvested by centrifugation and lysed mechanically with an emulsifier (Avestin). The GST-fusion protein was purified by GST affinity chromatography on a glutathione Sepharose column (Pharmacia). The N-terminal GST tag was then removed by overnight digestion with Prescission protease (GE Biosciences) at 4 °C. Cleavage with Prescission protease left 10 residues (GPLGSPEFPG) of the protease recognition site on the N-terminal of α-syn-Thr72Met. The α-syn-Thr72Met protein was separated from the GST tag and from the uncleaved fusion protein on a glutathione-Sepharose column.  , diabetes, and cataract surgery. The mother of the index case (I-2) was reported to be nearly bedridden with a tremor-predominant form of PD and severe cognitive impairment by the age of 80 years old. Information regarding the age at onset of her symptoms was not available. Her medical history included diabetes and arterial hypertension. One sister of the index case (II-2) presented at age 56 with memory problems and mild cognitive impairment on testing (MMSE 27/30 and ACE-R 73/100), but no signs of parkinsonism. Brain MRI showed bilateral frontal subcortical white matter lesions, which typically occur in the context of cardiovascular disease. Another sister of the index case (II-3) died at the age of 52 years due to an infectious disease. She was examined, and DNA was sampled at the age of 51 years; at that time, her clinical examination was normal.

Clinical reports
The pedigree of Family 2 (F2), originating from the region of Karaman in central-south Turkey, is shown in Fig. 1B. The index case (III-1) presented with a progressive hypokinetic-rigid parkinsonian syndrome, along with pyramidal signs, cognitive decline, and hallucinations at 39 years of age. Neurological examination revealed bradykinesia, rigidity, reduced arm swing, and altered postural reflexes, without tremor. The brain MRI, performed at the age of 41 years, showed no structural abnormalities. By the age of 46 years, the patient had become bedridden and dysphagic. In the past medical history, congenital deafness was reported. The maternal aunt of the index case (II-1) was also diagnosed with PD at the age of 55. At the age of 64, she showed dysphagia and hallucinations; 5 years later she became cognitively impaired. No other members of F2 were diagnosed with PD.
Of note, several members of F2 suffered from axonal sensorimotor neuropathy (Charcot-Marie-Tooth, CMT, type 2, HMSN2). The mother of the index case (II-2) suffered from HMSN2, which led to wheelchair dependence at the age of 70 years and later to the need for mechanical ventilation due to bilateral diaphragm paralysis. She also showed cognitive impairment at the age of 71 years. Whether signs and symptoms of PD were examined systematically in this individual remains unclear. Subject II-3 presented with HSMN2 at the age of 45 years, and with a pyramidal syndrome, increased muscle tone, and pseudobulbar symptoms a few years later. Brain imaging showed enlarged ventricles and cortical atrophy. Finally, subject III-2 was diagnosed with HMSN2 and cognitive impairment at the age of 43 but no signs or symptoms of PD were reported up to the age at last examination (46 years old). Brain atrophy predominantly in the cerebellum was seen in the brain MRI.

Genetic studies
In the index case of Family 1 (F1), Sanger sequencing revealed a heterozygous C/T transition in SNCA exon 4 (c.215C > T), predicted to lead to a threonine (Thr) to methionine (Met) amino acid change at codon 72 (p.Thr72Met). Screening for rare variants with coding or putative splicing effect in LRRK2 and GBA by Sanger sequencing as well as testing for copy number variants in SNCA, PARK2, PINK1, PARK7, ATP13A2, LRRK2 and GCH1 by MPLA were negative in this patient. Subsequent screening in F1 by Sanger sequencing showed the heterozygous SNCA c.215C > T (p.Thr72Met) variant in the other subject with overt parkinsonian phenotype (subject I-2), as well as in the subject II-3, who was free from PD symptoms and signs until she died at the age of 52 years, which was younger than the age of onset in the index case in this family. The SNCA variant was not present in the other examined and clinically unaffected members, II-2 and II-4. In

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Author Manuscript III-2, who were not reported with parkinsonian signs. Additional family members could not be tested for the SNCA p. Thr72Met variant. As several members in F2 suffered from HMSN2, the known neuropathy-causing genes were also tested. A heterozygous c. G2005T/ p.Glu669* disease-causing variant was identified in LRSAM1 (NM_138361) in the subjects II-1 and III-2. The index case with PD was also tested but did not carry the LRSAM1 variant.
Sanger sequencing of SNCA exon 4 in a series of 292 unrelated Turkish individuals detected no carriers of the c.215C > T variant. Haplotype analysis of the SNCA region in F1 and F2 revealed allele sharing among all the tested subjects carrying the p. Thr72Met variant for 4 markers (D4S2460, CGR784, D4S414, and CGR795) located closest to the SNCA gene ( Fig. 2), suggesting a common ancestor. We also estimated the frequency of the haplotype on which the SNCA variant likely originated by typing the same DNA markers in 56 individuals of Turkish origin (Fig. 2). Of note, for the D4S414 marker, closely flanking the SNCA variant, the allele linked to the variant in the PD patients was present in only 3.6% of the 112 tested chromosomes, further supporting the contention of a single common origin of the SNCA variant.

Protein studies
In protein aggregation studies, we observed that α-syn-Thr72Met began to aggregate robustly after 27 h of incubation, reaching maximum intensity at 45 h, while the aggregation of the wild-type protein remained relatively flat (Fig. 3A, and Supplementary Figure). Substituting in silico the threonine 72 (Thr72), which in the structure of the α-syn fibril (PDB ID: 6RT0) [13] is surrounded by three proximal valine residues, for methionine, we observed no steric clashes (Fig. 3B). Additionally, mapping the location of Thr72 on the tetrameric α-syn model of Wang et al. [12] reveals that Thr72 lies in the tetrameric interface (Fig. 3C).

Discussion
Here, we describe clinical, genetic, and functional studies in two families of Turkish origin with PD associated with the SNCA c.215C > T (p.Thr72Met) variant, which, to our knowledge, has not been reported before in patients with neurodegenerative disorders. the Turkish population. These results strongly suggest that the SNCA p. Thr72Met variant has originated from a single founder. Data regarding the presence and frequency of SNCA variants in the Turkish population are scarce. Two studies identified SNCA duplications in Turkish families [14,15], while another study failed to identify any SNCA variants [16]. This scarcity of data is not surprising, given the rarity of the SNCA variants in PD and related neurodegenerative disorders (an overview is provided in Table 1). Regarding the other missense SNCA variants identified in PD, only a very few can be considered as definitely disease-causing, while many others, detected in isolated cases or without convincing evidence of co-segregation with disease, remain of unclear significance ( Table  1). has not manifested signs of parkinsonism by the age of 46, but she suffered from cognitive impairment. Another mutation carrier (II-2) was reported with cognitive decline since the age of 71, without parkinsonian signs by the age of 73. However, the severe weakness due to the co-existing severe HMSN2 may have made it difficult to appreciate possible subtle signs of parkinsonism. Reduced penetrance has been extensively reported, especially for SNCA duplications but also in missense variants, while SNCA triplications appear to be more penetrant [7,[17][18][19][20][21][22]. The reasons for this incomplete penetrance and variable expressivity remain largely unknown. Multiple genetic and/or non-genetic modifiers might be involved [23,24], and much more work remains ahead to fill these important knowledge gaps.
An LRSAM1 c. G2005T/p.Glu669* variant was also identified in members of family 2 affected by HMSN2. LRSAM1 disruptive variants are an established cause of autosomal dominant axonal sensorimotor neuropathy [25]. Of note, in a previously published pedigree with HMSN2, three out of five affected members developed PD in addition to neuropathy [26], suggesting a possible link between LRSAM1 defects and the development of PD. In family 2, however, the LRSAM1 variant did not segregate with PD, as one of the two clinically affected parkinsonian cases (the index case, III-1) did not carry this variant. We, therefore, argue that the SNCA substitution is the PD-causing variant in these two Turkish pedigrees. The disease-causing SNCA missense variants reported so far are located in the amphipathic alpha-helical domain of α-syn, responsible for binding to lipid membranes. It has been considered that the aforementioned variants might exert their effects either by hampering the binding of α-syn to lipid membrane structures or by stimulating α-syn aggregation propensity, or both, but such mechanisms might not be shared by all of these variants [27]. In contrast, the p. Thr72Met variant is positioned in the central hydrophobic region of the α-syn protein, known as the non-amyloid-β component (NAC) domain. This domain is required for polymerization into amyloid filaments [28] and appears to be unique to α-syn among the synuclein family [29,30]. A viable hypothesis as to how the novel variant might cause PD would therefore be by enhancing α-syn aggregation potential. In our protein expression studies, we compared the aggregation kinetics of α-syn-Thr72Met with that of wild-type α-syn using a thioflavin-T aggregation assay. We observed that α-syn-Thr72Met began to aggregate robustly and much earlier than the wild-type (Fig. 3A). This result is not surprising as a substitution of a small hydrophilic sidechain (threonine) for one that is large and hydrophobic (methionine) is expected to result in significant structural alterations and thereby in changes in the aggregation propensity.
In order to understand potential reasons for the observed increase in aggregation propensity, we substituted in silico threonine 72 for methionine in fibrillar and tetrameric structural models of α-syn. We observed no steric clashes in the fibrillar model, with the hydrophobic sidechain of Met 72 potentially contributing to the stability of the hydrophobic pocket of the α-syn fibril (Fig. 3B). Additionally, mapping the location of Thr72 on the tetrameric α-syn model of Wang et al. [12] reveals that Thr72 lies in the tetrameric interface, and the substitution with the much larger methionine residue could potentially lead to destabilization of the complex (Fig. 3C).
In conclusion, we report a novel SNCA missense variant associated with PD in two Turkish families. Testing for this variant in patients with familial or sporadic PD of Turkish origins might detect additional carriers. Further functional analyses might offer new insights into the biochemical properties of p. Thr72Met and other PD-causing missense variants in α-syn, and to their mechanisms of action leading to neurodegeneration.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.