The New Mutation, E46K, of �-SynucleinCauses Parkinson and Lewy Body Dementia

Juan J. Zarranz, MD, PhD,1 Javier Alegre, MD,2 Juan C. Gomez-Esteban, MD,1 Elena Lezcano, PhD,1

Raquel Ros, PhD,2 Israel Ampuero, PhD,2 Lıdice Vidal, PhD,2 Janet Hoenicka, PhD,2 Olga Rodriguez, MD,3

Begona Atares, MD,4 Veronica Llorens, MD,5 Estrella Gomez Tortosa, MD, PhD,2,6

Teodoro del Ser, MD, PhD,7 David G. Munoz, MD, PhD,2 and Justo G. de Yebenes, MD, PhD2,6

Familial parkinsonism and dementia with cortical and subcortical Lewy bodies is uncommon, and no genetic defect hasbeen reported in the previously described sibships. We present a Spanish family with autosomal dominant parkinsonism,dementia, and visual hallucinations of variable severity. The postmortem examination showed atrophy of the substantianigra, lack of Alzheimer pathology, and numerous Lewy bodies which were immunoreactive to �-synuclein and ubiquitinin cortical and subcortical areas. Sequencing of the �-synuclein gene showed a novel, nonconservative E46K mutation inheterozygosis. The E46K mutation was present in all affected family members and in three young asymptomatic subjects,but it was absent in healthy and pathological controls. The novel mutation, that substitutes a dicarboxylic amino acid,glutamic acid, with a basic amino acid such as lysine in a much conserved area of the protein, is likely to produce severedisturbance of protein function. Our data show that, in addition to the previously described hereditary�-synucleinopathies, dementia with Lewy bodies is related to mutation of �-synuclein.

Ann Neurol 2004;55:164–173

Familial parkinsonism accounts for a significant pro-portion of cases of Parkinson’s disease (PD)1–3 and isrelated to several genetic defects that are inherited in adominant4–12 or recessive pattern.13–16 The genes re-sponsible for familial parkinsonism are not yet fullyknown, although several loci have been identified inthe last few years.4–16 The clinical phenotype presentedby patients with familial parkinsonism is variable. Notinfrequently, in addition to the typical parkinsoniansymptoms of PD, many of these patients present otherdeficits including dystonia, dysautonomia, cognitiveand behavioral changes, sleep disorders, and perceptivedeficits.

Dementia is not uncommon in patients with PD,both sporadic and familial. It was present in some pa-tients of the Contursi family, with the A53T mutationof �-synuclein4,17 and in families with mutations ofthe tau gene.18–21 In PD, whether sporadic or familial,the most common form of dementia is dementia withLewy Bodies (DLB), characterized by the presence ofLewy bodies in neocortical and paralimbic areas, andAlzheimer-type lesions.22–27 DLB is an heterogeneous

disorder with variable clinical and pathological featurestypically characterized by dementia, visual hallucina-tions, parkinsonism, and fluctuations in cognition andattention28,29 associated with the presence of Lewybodies in a pattern more widespread than that usuallyobserved in the brain nuclei of patients with PD. Theclinical diagnostic criteria of DLB proposed by an in-ternational consortium includes the presence of cogni-tive decline plus at least two of the following: (1) spon-taneous parkinsonian symptoms and signs, (2) visualhallucinations, and (3) fluctuations in consciousnessearly in the course of the disease.28,29 These criteriahave high specificity but low sensitivity for the diagno-sis of DLB, and the pathology remains the gold stan-dard for the diagnosis.30 Recent studies have suggestedthat the density of LBs in certain brain regions, such asthe parahippocampus and the cortical areas, correlateswell with the presence of dementia.27,30

The cause of DLB is likely to be related to multiplecauses. Most cases are sporadic but familial cases havebeen described.31–38 Genetic investigation of familialDLB has been very limited, and so far negative. Here,

From 1Department of Neurology, de Cruces Hospital, Departamentof Neuroscience, University del Paıs Vasco, Baracaldo, Vizcaya;2Banco de Tejidos para Investigaciones Neurologicas, Madrid; 3De-partment of Pathologic Anatomy, Hospital de Cruces, del PaısVasco University, Baracaldo, Vizcaya; 4Department of PathologicAnatomy, de Txagorritxu Hospital, Vitoria, Alava; 5Departament ofNuclear Medicine, de Cruces Hospital, Baracaldo, Vizcaya; 6Depart-ment of Neurology, Fundacion Jimenez Dıaz, Autonomous Univer-sity of Madrid; and 7Department of Neurology, Hospital SeveroOchoa, Leganes, Madrid, Spain.

Received May 27, 2003, and in revised form Aug 21. Accepted forpublication Aug 26, 2003.

Address correspondence to Dr Zarranz, Servicio de Neurologia, De-partamento de Neurociencias, Hospital de Cruces, Universidad delPaıs Vasco, 48903 Baracaldo, Vizcaya. Spain.E-mail: jjzarranz@hcru.osakidetza.net

ORIGINAL ARTICLES

164 © 2003 American Neurological AssociationPublished by Wiley-Liss, Inc., through Wiley Subscription Services

we report a family from the Basque Country with au-tosomal dominant parkinsonism, possible/probableclinical criteria, and typical pathological features ofDLB, produced by a novel mutation of �-synucleingene (SNCA). This mutation has not been found inthe control population or in other patients with famil-ial and sporadic parkinsonism, DLB, or dementia.

Patients and MethodsClinical Investigation of the Original FamilyA simplified version of the pedigree is presented in Figure 1.The hereditary pattern is definitely autosomal dominant. Forconfidentiality reasons, all members of this family are de-picted, regardless of their sex, by a diamond. The ancestorsof Patient I-1 are unknown. In the second generation, five ofeight siblings (Cases II-2, II-4, II-6, II-10, and II-15) wereaffected according to the family informants, and all of themhad transmitted the disease to their progeny. No medicalrecords from any of these possible affected members wereavailable. Case II-7 was a single woman without children andshe died asymptomatic at 65 years of age. Case II-8, an ob-ligate carrier of the mutation, died from cancer at 61 years ofage without neurological symptoms. Case II-12 died asymp-tomatic when he was 62 years old, and none of his childrenhave suffered the disease.

We have personally examined four of the seven affectedmembers in the third generation (Cases III-25, III-34, III-38, and III-39) and the only known symptomatic member ofthe fourth generation (Case IV-28). The clinical summariesof these five affected subjects are provided below.

Histopathological StudiesThe brain of the proband (Case III-34), extracted afterdeath, was immersed in formalin. After fixation 22 blockswere taken from representative cortical and subcortical areasaccording to standardized protocols.28 The paraffin-embedded sections were stained with routine histologicalmethods and processed for immunocytochemistry with anti-bodies against glial fibrillary acidic protein (1/5,000; Dako,Carpinteria, CA), phosphorylated neurofilaments (1/40;

Zymed, San Francisco, CA), tau (1/400; Sigma, St. Louis,MO), ubiquitin (Z0458,1/400; Dako), and �-synuclein (1/200, without formic acid pretreatment; Zymed).

Single-Photon Emission ComputedTomography StudiesAfter informed consent a single-photon emission computedtomography (SPECT)–FP-cit was obtained in Patient IV-28and in two asymptomatic children of the proband, aged 37and 41 years. A tomographic brain study with a Siemensdouble-head gamma-camera was performed after intravenousinjection of 185 MBq (2.5 mCi) of 123iodoflupane (2beta-carbomethoxy-3beta-[4-iodophenyl]-N-(3-fluoropropyl) nor-tropane (FP-CIT) as described.39

Clinical and Pathological Criteria for DiagnosisThe clinical diagnosis of DLB was made according to inter-national criteria28,29 that require the presence of progressivecognitive decline plus at least two of the following: (1) idio-pathic parkinsonism, (2) visual hallucinations, and (3) fluc-tuations in consciousness early in the course of the disease.The pathological criteria were those of the international con-sortium28,29 modified according to Harding and Halliday,30

namely, the presence of a high density of LBs, greater than3/200 magnification field in the parahippocampus, and theabsence of Alzheimer-type pathology.

The diagnosis of PD was made in accord with the recom-mendations of the London Brain Bank.40

Genetic StudiesInformed consent was obtained from patients, members oftheir families, and control subjects according to the ethicalrequirements of the Clinical Research Committee, Funda-cion Jimenez Dıaz, Madrid. Genomic DNA was isolated ei-ther from peripheral leukocytes by standard phenol/chloro-form and ethanol precipitation methods or from embeddedbrain tissue adding xylene to remove the paraffin and usingQIAamp DNA Mini Kit (Qiagen, Chatsworth, CA). Poly-merase chain reaction (PCR)–based methods were used forthe molecular analysis.39 Park1 exons were amplified using

Fig 1. A fragment of the pedigree of the family with dementia with Lewy Bodies caused by the E46K mutation of �-synuclein(hatched symbol: affected by history; filled symbol: involvement confirmed by personal examination; slashed symbol: deceased).The true order of birth and sex is not shown to preserve confidentiality.

Zarranz et al: New �-Synuclein Gene Mutation 165

the oligonucleotides primer pairs described in Table 1. Someof these primers were selected according to previous re-ports6,8 and others according to National Center for Biotech-nology Information database (www.ncbi.nlm.nih.gov).

Amplification reactions were performed by means of Am-pliTaq Gold DNA polymerase (Perkin-Elmer, Applied Bio-systems Division, Foster City, CA) in a final volume of 25�lby use 250ng of genomic DNA. The MgCl2 concentration(1.5–1.75mM) and annealing temperature (50–55°C) wereoptimized when necessary. After initial denaturation at 94°Cfor 10 minutes, amplification was performed through 35 cy-cles of 94°C for 30 seconds, the annealing temperature for30 seconds, and 72°C for 45 seconds, for extension. After afinal 10-minute extension at 72°C, the amplified products ofPark1 exons 2 and 3 (previously referred to as 3 and 46,8)were subjected to direct sequencing in the index case withthe same primers used for amplification, using afluorescence-based automated sequencing technique (AppliedBiosystem 373a DNA sequencer, Perkin-Elmer Model). Ex-ons 4 and 5 and coding region of exon 6 of the Park-1 genewere screened by single-strand conformation polymorphismanalysis using the Genephor System (Amersham, PharmaciaBiotech, Sweden) at both 5 and 15°C according to the man-ufacturer’s recommendations. PCR products were digestedusing StyI enzyme (New England Biolabs, Beverly, MA) inaccordance with the manufacturer’s protocols. After diges-tion, the fragments were electrophoresed on a 12% nonde-naturing polyacrylamide gel at 150V for 2 hours and visual-ized by silver staining with the PlusOne DNA Silver StainingKit (Amersham Pharmacia Biotech).

Selection of Controls and Other Clinical CasesIn addition to the original family, we included seven DLBfamilies from different regions of Spain, 22 sporadic DLBcases (20 of them confirmed by pathology), 34 patients withother neurological disorders, and 213 healthy controls fromblood banks.

ResultsRetrospective Clinical DataWe questioned the relatives of the eight deceased caseswhom we had not personally examined about their

clinical picture, namely, the presence of tremor, dis-ability, visual hallucinations, or dementia. The re-sponse to L-dopa could not be evaluated because allhad died before this drug was available. Age at onset ofthe disease ranged from 50 to 65 years, and age atdeath from 64 to 75 years. One patient had died earlyat age 63 years from cancer. Mild tremor at rest waspresent in approximately 50% of the patients. All thepatients became wheelchair or bed bounded. Evalua-tion of dementia was difficult because of severe mutismand immobility but was probable as a late event in fourof the eight cases. Two patients had presented vividvisual hallucinations.

Report of Representative CasesPATIENT 1 (PROBAND, CASE III-34). Patient 1 was a 69-year-old man, who had noticed tremor in his right ex-tremities, rigidity, generalized bradykinesia, and mem-ory loss for 2 years before his first consultation andagitated nocturnal sleep for a long time. Examinationshowed a normal mental status with hypophonicspeech, hypokinesia and rigidity in both arms, moresevere in the right side, together with tremor at rest inthe right hand. He had normal gait, balance, and co-ordination. The polysomnography showed a drastic re-duction of sleep time, with only short periods of phaseI of nonrapid eye movement sleep and absence of rapideye movement sleep (detailed polysomnography resultswill be reported elsewhere). Blood counts and chemis-tries were normal. Brain magnetic resonance imagingshowed nonspecific changes. The patient improvedwith L-dopa and selegiline but developed mild choreicdyskinesia and cognitive decline 2 years later. He wasdisoriented in time and place, his Mini-Mental StateExamination was 16 of 30, his alertness fluctuated, andhe had delusions, visual hallucinations that improvedwith risperidone, and confusion of reality. One yearlater, he had unexplained sudden falls. In January2002, 8 years after disease onset, dementia was severe

Table 1. PCR Primers for Exonic Amplification of �-Synuclein

Exon Primers 5�/3�, Forward and Reverse ReferenceProductSize (bp)

2 5�-TCAAAGTGTGTATTTTATGTTTTCC-3� Kruger and Colleagues8 1975�-CAAACTGACATTTGGGGTTTAC-3�

3 5�-GCTAATCAGCAATTTAAGGCTAG-3� Polymeropoulos and colleagues6 2185�-GATATGTTCTTAGATGCTCAG-3�

3bisa 5�-GCTAATCAGCAATTTAAGGCTAG3� Designed by the authors 1485�-CACTAGATACTTTAAATATCATC-3�

4 5�-GTCACTGTTATTCTACCACC-3� Designed by the authors 2595�-GAACCGTAATCTCACCAGC-3�

5 5�-GGTGTGCCATTTTCAAGATC-3� Designed by the authors 2655�-GACAGGTTTTTGGTACTGTG-3�

6 5�-GCCATTTCCTATCTCATTGG-3� Designed by the authors 1845�-CTTGTACAGGATGGAACATC-3�

aPrimers used for amplification of exon 3 from DNA obtained from brain tissue.

166 Annals of Neurology Vol 55 No 2 February 2004

and the patient was incontinent. He died of pneumo-nia in February 2002. An autopsy was performed.

In summary, this man presented with nocturnal ag-itated insomnia for more than 10 years followed by ageneralized parkinsonian syndrome with mild memoryimpairment for 7 years and a severe cognitive declineand hallucinations for 2 years.

PATIENT 2 (CASE III-25). Patient 2, a 67-year-oldwoman, was seen in November 1992 because of a2-year history of bradykinesia for hand movements,and postural abnormalities with flexion of the trunk,and festination of gait. The patient was amimic, trem-orless, with a marked flexion of the trunk, severe aki-nesia, and rigidity in the four limbs. Gait was slow butunaided, without arm swing but at a normal pace. Bal-ance was preserved. She improved functionally withL-dopa and selegiline. Two years, later the patient de-veloped memory problems, and when the dose ofL-dopa was increased, mental confusion and visual hal-lucinations appeared. Her cognitive and motor deficitsworsened progressively, and she died dependent fordaily living activities at age 73 years.

PATIENT 3 (CASE III-38). Patient 3 was a 58-year-oldwoman who developed generalized bradykinesia withearly flexion of the trunk and without tremor. Her mo-tor deficits became worse during the following years de-spite treatment with L-dopa, bromocriptine, and selegi-line. We examined her when she was 69 years old, and,in addition to her parkinsonian syndrome, she hadfluctuating behavioral changes, mental confusion, disori-entation, diurnal hypersomnia, confabulations, visualhallucinations and frequent falls. A neurological exami-nation confirmed a fluctuating mental status.

Changes of antiparkinsonian medication providedno benefit. After a hip replacement, her mental statusdeteriorated, she became confused and delirious, withrestless sleep, confined to a wheelchair or bed, mute,and demented. She died in 2001.

PATIENT 4 (CASE III-39). Patient 4 was a 60-year-oldwoman who reported bradykinesia that prompted herearly retirement. She was diagnosed as having PD andtreated with L-dopa. The effects of the treatment areunknown but eventually she became unable to walk ataround age 70 years. Two years later, her speech wasunintelligible. She was placed in a nursing home at age74 years, dependent for daily living activities [invalid]and demented, and she is still alive at age 78 years. Shedoes not recognize her relatives or nurses, is double in-continent, and wheelchair bound. L-Dopa, 1,000mg/day, does not modify her motor deficits now. She ismoderately amimic. Spontaneous and automatic ocularmovements are normal. Speech is unintelligible and sheonly produces some automatic utterances. She can

obey only inconsistently some simple commands (eg,raise your hand) or perform pronosupination move-ments of the hands. A mild postural tremor is presentin both arms, slightly more marked on the left. Stanceand walking are impossible.

PATIENT 5 (CASE IV-28). Patient 5 was a 50-year-oldman who was seen in May 2001 for a 1-year history ofdiffuse discomfort and muscle rigidity in his right arm,shoulder, and lateral cervical region. He noticed a reduc-tion of right arm swing during walking, and loss ofmanual dexterity for skillful movements such as writing,shaving, or using a computer mouse. He had no tremor.He felt sad and depressed and left his job transiently.

The neurological examination disclosed amimia, re-duced blinking and fixed stare, normal ocular move-ments, and hypophonic and monotonous speech. Hehad marked bradykinesia and moderate rigidity, andno tremor in the right limbs. Stretch reflexes and plan-tar responses were normal. He walked with a milddragging of the right leg and reduced right arm swing.Balance was normal but some slowing of postural re-flexes was observed. Mental status was considered nor-mal. Routine blood and urine analyses and brain mag-netic resonance imaging were normal. Treatment withselegiline and pergolide improved his motor and affec-tive deficits and he resumed his job.

At the beginning of 2003, he scored 27 and 20 inthe United Parkinson’s Disease Rating Scale–III in“off-drugs” on 2 consecutive days. A mild resting andpostural tremor appeared in the upper extremities.Apomorphine, 2mg SC, reduced the motor score by30%, and by 59% after a second challenge with 3mg.L-Dopa, 250mg PO, reduced the United Parkinson’sDisease Rating Scale–III score by 15%, and by 57% ata dose of 375mg. A comprehensive neuropsychologicalexamination gave results within normal limits, al-though the patient had some difficulties in learning thebases of classification in the Wisconsin Card Sortingtest. He provided 39% of wrong answers, 19.5% dueto perseveration, a score still normal but below themean for his age and education. This result was con-sidered suggestive of mild impairment in cognitive flex-ibility. A SPECT-FP-cit showed a clear reduction ofnigrostriatal dopaminergic function on the left striatummore marked in the putamen, contralateral to the moresymptomatic right limbs (Fig 2).

Pathological FindingsThe macroscopic external examination of the brain wasunremarkable. The brain weight after fixation was1,400gm. A moderate pallor of the substantia nigraand locus coeruleus was observed. The main micro-scopic finding with the routine histological stains was amoderate to severe neuronal loss, microspongiosis, freeand phagocyted neuromelanin, and reactive astrogliosis

Zarranz et al: New �-Synuclein Gene Mutation 167

in the substantia nigra. Some neuronal loss was presentin locus coeruleus and was more severe in the dorsalmotor nuclei of the vagus nerve. With the hematoxylinand eosin stain, abundant Lewy bodies of the classictype were observed in the substantia nigra, basal nu-cleus of Meynert, perifornical area, medullary and pon-tine tegmentum, periaqueductal gray matter, raphe nu-clei, locus coeruleus, area postrema, and dorsal motor

nuclei of the tenth nerve. Some pale bodies and axonalspheroids were present in the same structures. Manynonconcentric and some concentric LBs were observedin the anterior cingulated cortex (Fig 3B, C). Somebizarre inclusions (elongated or serpiginous) were de-tected in the pontine (see Fig 3D) and bulbar tegmen-tum and in the dorsal motor nuclei of the vagus nerve.Some scattered amyloid nonneuritic plaques were ob-

Fig 2. Single-photon emission computed tomography (SPECT)–FP-cit. The findings are within normal limits in one control and inone asymptomatic carrier of the mutation (A, B). The dopaminergic transport is reduced in both lenticular nuclei, more on the leftside in Patient IV-28 (C), congruent with the asymmetric clinical picture, more severe on the right side.

Fig 3. Summary of the pathological findings in the index case. (A) Substantia nigra. Severe neuronal loss, secondary spongiosis, andpigment laden macrophages. (B, C) Cingular cortex. Two Lewy bodies, one concentric and one nonconcentric. (D) Pontine tegmen-tum. An atypical elongated rod-like Lewy body. (E) Locus caeruleus. Classic Lewy body. Hematoxylin and eosin used for all panelsshown.

168 Annals of Neurology Vol 55 No 2 February 2004

served in the frontal neocortex. There were no plaques,tangles, or granulovacuolar degeneration in the hip-pocampal formation. There were no other relevant mi-croscopic abnormalities with the routine stains in cor-tical or subcortical structures.

The immunocytochemical methods against ubiquitinand �-synuclein confirmed the presence of Lewy bod-ies in the above-mentioned structures (Fig 4). In addi-tion, more axonal spheroids and many dystrophic neu-rites (Lewy neurites [LNs]) positive for ubiquitin and�-synuclein were observed. A semiquantitative distribu-tion of LBs, LNs, and axonal spheroids is presented inTable 2. In the hippocampal formation, there was amoderate to abundant deposit of LBs and LNs in thepyramidal layer (in all the sectors), but there was noimmunoreactivity in the granular layer. The amyloidplaques in the neocortex showed a tiny granular stain-ing for ubiquitin but were negative for tau immunore-activity. There were no plaques or tangles in any area.

Molecular FindingsSequence genomic DNA analysis of the different exonsof Park1 gene of the index case failed to show any ofthe previously reported mutations but disclosed a singlebase pair change at position 188 from G to A (G188A)

relative to the published GenBank ID L08850 (Fig 5).G188A transition replaces the acidic polar glutamicresidue with the basic polar lysine residue at position46 (E46K) and abolishes a StyI restriction site (see Fig6). Mutation screening for the G188A change in thisfamily showed complete segregation with the DLBphenotype with the exception of the carriers of thismutation that are asymptomatic. Such individuals areyounger than the other affected members. We per-formed haplotype analysis in the available members ofthe family using three microsatellite markers thatmapped around the Park1 gene. This analysis identi-fied a haplotype shared by E46K carriers (data notshown). We excluded the presence of another geneticlesion in addition to E46K by analysis of single-strandconformation polymorphism of the rest of the Park1coding exons. We did not observe any band shift.

The search for the G188A change in 276 Spanishhealthy and pathological controls was negative.

DiscussionClinical and Pathological FindingsThe patients described here presented autosomal dom-inant severe parkinsonism with dementia, recognized in

Fig 4. Summary of the immunocytochemical findings in the index case. (A) Substantia nigra. Typical Lewy body (LB) inclusion.(B) Substantia nigra. Axonal spheroid. (C) Frontal neocortex. Some isolated LBs were found. (D) Anterior cingulate cortex. (E)Amigdaloid nucleus. Many LBs and Lewy neurites fulfilling criteria for neuropathological diagnosis of dementia with LBs.�-Synuclein immunocytochemistry was used in all panels.

Zarranz et al: New �-Synuclein Gene Mutation 169

most patients 2 or more years after disease onset, withlate hallucinations at therapeutic doses of L-dopa, andfluctuations in the level of consciousness. The clinicalfeatures of these patients were more consistent withmotor than with perceptive or cognitive deficits, atleast in the initial stages of the disease. Cognitive de-cline, hallucinations, and fluctuations of consciousnessappeared eventually in most but not all patients. Thepathological findings were unambiguously consistentwith DLB, because there was a high density of LBspresent not only in the subcortical nuclei but also inthe parahippocampus, amygdala, and cortex. NoAlzheimer-type pathology was present.

Several clinicopathological syndromes such as DLB,pure autonomic failure, and behavioral disorder ofrapid eye movement sleep share the same histologicalmarker with PD, namely, the LB, but with differentregional and cellular distribution. In these syndromes,the clinical symptoms are poor predictors of the pa-thology,30 and, in general, DLB is seen more fre-quently as a pathological finding than as a clinical di-agnosis

It is not clear how these syndromes with their dif-ferent clinical phenomenology are related to synucleinpathology. In the absence of a proven common cause,the relationship between these syndromes, particularlybetween PD and DLB, is a matter of controversy dueto clinical and pathological overlap. Approximately30% of PD patients eventually develop dementia, andnumerous LBs are found in their cortical and subcor-tical brain areas.24 On the other hand, a parkinsoniansyndrome associated with cognitive impairment is amain clinical diagnostic criterion for DLB, and, insuch cases, LBs are regularly found at autopsy in thesubstantia nigra. A consensus conference held in 1996concluded that for pragmatic reasons it was appropri-ate to maintain PD and DLB as different clinicopath-ological syndromes.28 They suggested taking an arbi-trary 12-month interval (later expanded to 24months) between the beginning of motor symptomsand the cognitive decline as indicative of DLB,whereas they reserved the denomination of PD withdementia (PDD) for those cases in which the demen-tia occurs later in the course of the disease.29 Themain reason for such a distinction is that, in PDDpatients who develop dementia after many years ofevolution, a confounding comorbidity with Alzheimerpathology is common.24,25 There are, however, pa-tients with DLB who develop dementia more than 2years after disease onset.

Fig 5. Genomic DNA sequencing corresponding to exon 3 of Park1 gene of the index case of this family showed a single base pairchange at position 188 from G to A (G188A) that produces a nonconservative mutation, E46K, of the �-synuclein protein.

Table 2. Summary of the Distribution of the MainNeuropathological Lesions

StructureLewy

BodiesLewy

NeuritesAxonal

Spheroids

Substantia nigra ��� ��� ��Locus caeruleus �� � �N. motor dorsal X �� �� �Tegmentum medulla �� �� 0Tegmentum pons �� �� 0Raphe nucleii � � �Amygdala ��� ��� �Hippocampus �� ��� 0Parahippocampus ��� �� 0Cingular cortex ��� �� 0Temporal neocortex �� �� 0Frontal cortex � 0 0Parietal cortex � 0 0Occipital cortex 0 0 0Thalamus � �� 0Putamen 0 0 0Globus pallidus 0 0 0Basal n. Meynert �� � 0Hypothalamus �� �� 0Cerebellum 0 0 0

0 � lesion absent; � � scattered lesion; �� � moderately abun-dant lesions; ��� � abundant lesions, more than three in �200microscopic field.

170 Annals of Neurology Vol 55 No 2 February 2004

The E46K Change Is a Pathogenic MutationResponsible for Dementia with Lewy Bodies,Not a PolymorphismThe E46K change is a pathogenic mutation responsiblefor DLB in this family and not a polymorphism for thefollowing reasons. (1) It cosegregated with the presenceof clinical symptoms with the exception of the asymp-tomatic gene carriers who had not yet reached the ex-pected age of onset. (2) It is not present in the controlpopulations. (3) It involves an area of �-synuclein thatis well preserved during evolution. (4) It involves anarea of �-synuclein that is critical for the protein func-tion. (5) It produces a nonconservative change of po-larity in that area of the protein, substituting a dicar-boxylic amino acid, glutamate, with a basic amino acidin one of the imperfect repeats KTKEGV, near theamino terminus, that are critical for the protein func-tion.

The first Park1 mutation, A53T, consists in thetransversion at the nucleotide position 209 from gua-nine to adenine, resulting in the change of alanine tothreonine in the mutant peptide. This mutation wasfound in a large Italian family and several Greek fam-ilies.6 The other mutation in Park1, A30P, was iden-tified in a PD German family and consists in the sub-stitution of guanine to cytosine at position 88 of thecoding sequence, causing the change of alanine to pro-line.8 These patients have typical L-dopa–responsiveparkinsonism except for a relatively early age at onset.4

There is, however, phenotypic variability among pa-tients, because, in the Contursi kindred, three of fivepatients whose clinical data were reported had demen-tia and one had visual hallucinations. Surprisingly, arecent review of the neuropathology in one of thesebrains reported that the density of LBs in the cortexwas low but that there was extensive tau pathology,41

not present in our case.

The Mutation Now Described Greatly Interferes withProtein FunctionLewy bodies are mainly composed of misfolded�-synuclein, a presynaptic protein of 140 amino acidsthat is abundant in the brain.42 An important charac-teristic of the �-synuclein primary structure is the pres-ence within the first 95 residues of imperfect repeatsthat bind synthetic unilamellar vesicles rich in acidicphospholipids, suggesting that it may be a lipid bind-ing protein.42 Interestingly, both the A53T and theA30P lie in the repeat region of the molecule, and,although the mechanistic pathogenetic basis is notclear, it probably involves novel functions of the mu-tant filamentous �-synuclein.41 There is an altered ex-pression of synucleins in Lewy bodies, with increasedlevels of �-synuclein mRNA and reduced levels ofsynuclein.43

Transgenic mice expressing wild-type human�-synuclein present �-synuclein and ubiquitin-immunoreactive intracytoplasmic and intranuclear in-clusions in their cortical neurons, fewer dopamine ter-minals in the striatum, and motor deficits.44 Inaddition to its nuclear and cytoplasmic function,�-synuclein plays a role in the regulation of dopa-mine release at the presynaptic terminal. Mice lacking�-synuclein had an increased liberation of dopa-mine.45 Human mesencephalic lines transfected withthe A53T mutation present a reduced KCl- and anincreased amphetamine-induced release of dopamineand accumulation of this neurotransmitter in the cy-tosol where dopamine could trigger excessive produc-tion of free radicals and mitochondrial dysfunctionleading to cell death.46 The mutation now reportedlies also in one of these imperfect repeats, but theradical change that it produces in the protein greatlymodifies the polarity of the region. The impact ofthis change in protein function therefore is predicted

Fig 6. Restriction analysis in members of the family. Numbers refer to the position of each individual in the pedigree as shown inFigure 1. Subject III-31 has surpassed the usual age of onset. Subjects IV-15 and IV-17 are not at risk of heredity. Subjects IV-24,IV-25, IV-26, IV-48, and IV-50 are at risk. Subject III-34, III-39, and IV-28 are symptomatic. The exon 3 of Park1 gene wasamplified by polymerase chain reaction (PCR) and digested using StyI enzyme. The G188A mutation abolishes a restriction site.(single asterisk) Molecular weight marker. (double asterisks) Index case PCR product (not digested). The PCR products of the restof the patients produced a band with the same weight when electrophoresed (not shown).

Zarranz et al: New �-Synuclein Gene Mutation 171

to be greater than those more conservative changesalready described in other residues, and that could ex-plain a more widespread distribution of �-synucleinpathology in the cases described here than in the pa-tients with the mutations already reported.

Our Findings Help to Include Dementia with LewyBodies in the Spectrum of Parkinson’s DiseaseBecause different mutations of the same gene couldproduce variable clinical phenotypes and with gradualseverity of the pathology, our findings support a closerelationship between DLB and PD. Both PD and DLBare caused by �-synuclein pathology. Other familialcases of PD or DLB could be caused by other muta-tions of �-synuclein. Therefore, in families with auto-somal dominant PD or DLB, a full search for muta-tions in the Park-1 gene is warranted.

Synucleinopathies, both genetic and sporadic, alsocould be related to mutations of other genes that codefor proteins interacting with �-synuclein in the case offamilial disease, or to postranslational alterations of thisprotein in sporadic cases. Gwinn-Hardy and col-leagues47 recently reported a family with patients af-fected by diseases ranging from Parkinson’s disease todementia and psychosis linked to chromosome 4p15, adifferent locus from that of the �-synuclein gene. Theyalso had widespread synuclein pathology, from pleo-morphic Lewy bodies to synuclein-positive glial inclu-sions and dystrophic neuritis. We recently have re-ported a case of pathologically confirmed progressivesupranuclear palsy with hyperphosphorylation of pro-tein tau associated with a mutation of the Park-2 gene,which occurred in a patient with a haplotype of risk fora tauopathy.48 In sporadic PD patients with�-synuclein pathology, postranslational changes may berelated to several mechanisms such as nitric oxide–relatedoxidation of tyrosine residues of �-synuclein,49 abnormalaggregation related to excess of free radicals or inhibitionof mitochondrial respiratory chain,50 abnormal�-synuclein phosphorylation,51 and other putative alter-ations that should be investigated.

Our study provides relevant data for understandingthe pathogenesis of DLB. The investigation of the roleof �-synuclein and the effects of the new mutated mol-ecule in cell function will provide the clues for a betterunderstanding of the molecular mechanism of thesedisorders and for developing rational neuroprotectivestrategies.

This work was funded by a grant from the Progressive SupranuclearPalsy Society (06.2001) and by the Areces Foundation, (03-2000,J.G.d.Y.)

We thank A. Espana for technical support with the molecular stud-ies and C. Varela for her help with the inmunohystochemical pro-

cedures. We are also grateful to A. Molano for the neuropsycholog-ical evaluation of Patient IV-28.

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