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Journal of NeurologyOfficial Journal of the EuropeanNeurological Society ISSN 0340-5354Volume 258Number 11 J Neurol (2011) 258:2043-2047DOI 10.1007/s00415-011-6066-1

Presenilin 2 mutation R71W in an Italianearly-onset sporadic Alzheimer’s diseasecase

Paola Piscopo, Giuseppina Talarico,Lorenzo Malvezzi-Campeggi, AlessioCrestini, Roberto Rivabene, MarinaGasparini, Giuseppe Tosto, et al.

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ORIGINAL COMMUNICATION

Presenilin 2 mutation R71W in an Italian early-onset sporadicAlzheimer’s disease case

Paola Piscopo • Giuseppina Talarico • Lorenzo Malvezzi-Campeggi •

Alessio Crestini • Roberto Rivabene • Marina Gasparini • Giuseppe Tosto •

Nicola Vanacore • Gian Luigi Lenzi • Giuseppe Bruno • Annamaria Confaloni

Received: 4 November 2010 / Revised: 11 January 2011 / Accepted: 19 April 2011 / Published online: 5 May 2011

� Springer-Verlag 2011

Abstract Mutations in the presenilin 2 (PSEN2) gene are

less commonly identified as genetic causes of early-onset

familial Alzheimer’s disease than mutations in the amyloid

precursor protein (APP) and the presenilin 1 (PSEN1)

genes. In fact, only 23 different mutations in the PSEN2

gene have been described in the literature. This paper deals

with a sporadic case of a 55 year-old subject bearing an

amino acid substitution from arginine to tryptophan at

codon 71 of PSEN2 and presenting a peculiar early-onset

Alzheimer’s disease phenotype.

Keywords Genetics � Mutation � Presenilins �Early-onset Alzheimer’s disease

Background

Autosomal dominant early-onset Alzheimer’s disease (AD)

is genetically heterogeneous. Approximately half of these

AD cases are associated with a mutation in the presenilin 1

(PSEN1) gene, whereas mutations in the presenilin 2

(PSEN2) and the amyloid precursor protein (APP) genes are

less frequently found. Mutations in PSEN2 on chromosome 1

were first described in 1995 and are rare causes of familial

Alzheimer’s disease [1]. Recently, particular attention was

dedicated to describing the phenotypic expression of PSEN2

mutations [2]. So far, 18 pathological PSEN2 mutations have

been reported (http://www.molgen.ua.ac.be/ADMutations/).

These mutations share some common phenotypic features,

even though some clinical and biological features were fre-

quently described as being particular to a certain mutation

[3–8]. Here, we describe a subject bearing the PSEN2 R71W

mutation and presenting a peculiar early-onset sporadic

Alzheimer’s disease phenotype.

Materials and methods

The patient was enrolled at the Memory Clinic of the

Department of Neurology and Psychiatry, and informed

consent for blood samples for the genetic study was

obtained not only from the patient but also from his two

children prior to their inclusion in the study. This study was

approved by the local Institutional Ethics Committee (in

accordance with the ethical standards of the Helsinki

Declaration). Venous blood was drawn in EDTA from all

subjects. The DNA was isolated from blood cells by the

standard method. The exonic regions 4–13 of PSEN1, 3–12

of PSEN2 and 16–17 of APP genes were PCR amplified by

specific primers [9]. Briefly, amplified DNA was carried

out in 20 ll reaction volumes containing: 200 ng of

genomic DNA, 19 buffer, 2.5 mM MgCl2, 10% DMSO,

0.2 mM of each dNTP, 1 pmol/ml of each primer, and

0.2 ll AmpliTaq Gold (Applied Biosystems, Carlsbad,

California, USA). The PCR cycling conditions were: one

cycle at 94�C for 10 min; 35 cycles at 94�C for 1 min,

P. Piscopo � L. Malvezzi-Campeggi � A. Crestini � R. Rivabene

� A. Confaloni (&)

Department of Cell Biology and Neurosciences,

Istituto Superiore di Sanita, Viale Regina Elena, 299,

00161 Rome, Italy

e-mail: annamaria.confaloni@iss.it

G. Talarico � M. Gasparini � G. Tosto � G. L. Lenzi � G. Bruno

Department of Neurology and Psychiatry,

University of Rome ‘‘Sapienza’’, Rome, Italy

N. Vanacore

National Centre for Epidemiology,

Surveillance and Health Promotion,

Istituto Superiore di Sanita, Rome, Italy

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J Neurol (2011) 258:2043–2047

DOI 10.1007/s00415-011-6066-1

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60�C for 1 min, 72�C for 1 min; and a final step at 72�C for

10 min. Then direct sequencing was performed in both

strands using the DNA sequencer Beckman CEQ 8000 and

then analysed using the CEQ 2000 software (Beckman

Coulter, Inc. Brea, CA, USA). The 12 coding exons of

PGRN and exons 9, 10, 12, and 13 of TAU were also

analysed. The genotyping of APOE was performed through

PCR amplification and HhaI restriction enzyme digestion.

Phylogenetic analysis

A comparison of PSEN2 gene sequences by means of the

multiple sequence alignment was performed using the

CLUSTALW program (http://www.ebi.ac.uk/clustalw). We

used PolyPhen software (http://genetics.bwh.harward.edu/

pph/) for the impact prediction on the PSEN2 structure and

function of the amino acid substitution.

Clinical features

The proband was a 55 year-old subject, who, at 53 years of

age, developed insidious memory loss, impairment of

shifting abilities, and behavioural disturbances such as

irritability and anxiety. The family history was negative for

neurodegenerative disease and positive for diabetes. His

past medical history was significant for blood hypertension

and hypertrigliceridaemia. At presentation, the neurologi-

cal examination was normal except for the presence of

primitive reflexes. The Mini Mental Status Examination

(MMSE) score was 29/30. Formal neuropsychological

testing showed impairment in verbal and visual recall and

the slowing down of selective attention (Table 1). Brain

MRI revealed bilateral, slight but diffuse cortical atrophy

(Fig. 1); hypoperfusion in temporal and parietal regions

was present on 99Tc-HM-PAO single photon emission

Table 1 Cognitive performances

Test Cut-off scores*

MMSE 29/30 [24

Rey 15-word list

Short-term recall 28.80 C28.53

Long-term recall 3.70 C4.69

Recognition 9 14.1 ± 1.1

RCF

Copy 31 C23.47

Immediate recall 3 C6.44

Delayed recall 0 C6.33

TMT

A-form 22 s \94 s

B-form 169 s \283 s

Stroop cw (t-score) 33 C35

CPM 24.8 C18.96

F.A.S. 33.8 C17.35

Animal naming 3 [8

Naming test 75 C61

Rey 15-word-list verbal memory and learning, Rey complex figure(RCF) constructional ability and visual memory, Trail making test(TMT) conceptual tracking and shifting, Stroop cw selective attention,

Coloured progressive matrices (CPM) abstract thinking, F.A.S. andanimal naming phonemic and semantic fluency

* All raw scores were adjusted for demographic variables and com-

pared with cut-off scores according to normative data for the Italian

population [11, 12]

Fig. 1 MRI analysis of the proband. The RMI study was performed

at time of AD diagnosis (a) and after 5 years (b). The image in

a shows a bilateral diffuse cortical atrophy that is present without

important changes in b

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computed tomography (SPECT) (Fig. 2). The proband

satisfied DSM-IV and Alzheimer’s disease and related

disorders association (NINCDS-ADRDA) criteria for

probable Alzheimer’s disease and treatment with AChEI

(acetylcholinesterase inhibitors) rivastigmine and SSRI

(serotonin-specific reuptake inhibitor), citalopram was

started. Two years later the MMSE score was 23/30 and

agitation and compulsive behaviour had developed:

risperidone therapy was initiated while rivastigmine was

discontinued due to its poor effectiveness.

Results

Genetic analysis of the proband’s PSEN1, APP, TAU and

PGRN genes disclosed no abnormalities, whereas analysis

of the PSEN2 gene revealed a missense mutation that

showed a cytosine to thymine transition in exon 4 leading

to a predicted amino acid change from arginine to trypto-

phan at codon 71 in heterozygosis. This sequence variant

was localized at the N-terminal region of the PSEN2 pro-

tein (Fig. 3). The R71W substitution was identified in the

proband and in his two sons. One of them was a 31 year-

old subject who presented with mental retardation and

generalized seizures following neonatal hypoxia. The other

30 year-old sibling showed a normal cognitive profile. The

identified mutation was not found in a cohort of 247

unrelated individuals (117 healthy subjects and 130 AD

patients), indicating that it is not a common polymorphism.

In silico phylogenetic analysis of the PSEN2 sequence

showed that the arginine residue was not evolutionarily

conserved with a normalized conservation score of ?1.4

(by ConSeq Software). However, further computational

analysis using the PolyPhen server, a sequence/structure-

based tool for predicting the functional effect of mutations,

showed that the tryptophan substitution had never been

found during evolution. Therefore, the PSEN2 R71W

variant is possibly damaging with a position-specific

independent count (PSIC) score difference of 2.228, sug-

gesting that the mutation may disrupt protein function and

structure. Regarding the ApoE genotype, all of the studied

subjects were e3/e3.

Discussion

This study reports the identification of a sporadic AD

subject bearing the PSEN2 R71W mutation and presenting

a peculiar phenotype characterized by early onset and

behavioural symptoms. The R71W variant identified on

Fig. 2 SPECT of the proband.

The image shows the result of

(99 m) Tc-HM-PAO SPECT

performed 1 year after the AD

diagnosis. The study shows a

hypoperfusion in right temporal

and parietal regions

J Neurol (2011) 258:2043–2047 2045

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exon 4 of PSEN2 predicts an arginine-to-tryptophan sub-

stitution in the protein sequence. The arginine residue

at codon 71 of PSEN2 is not evolutionarily conserved;

however, the arginine-to-tryptophan substitution could

effectively alter the protein structure/function; in fact,

tryptophan homologues of PSEN2 were not identified

during evolution as assessed by in silico analysis. This

mutation was described for the first time by Guerreiro et al.

[5]. They defined this mutation as possibly pathogenic,

following an algorithm key to classify the mutation’s

pathogenicity. Interestingly, Guerreiro et al. identified the

mutation in an elderly sporadic Alzheimer’s case, whereas

our patient was an early onset case and the mutation was

inherited by the two sons, both bearing e3/e3 ApoE geno-

type. Moreover, it is important to underline the fact that

neuroimaging data showed some peculiar differences

between Guerreiro’s patient and our patient. In fact, in the

clinical case of Guerreiro’s, brain MRI demonstrated a

chronic periventricular microangiopathic leukoencepha-

lopathy with multiple nucleo-capsular and periventricular

white matter lacunar infarctions, while in our case, a

bilateral, diffuse, slight cortical atrophy was present.

A bilateral hypoperfusion in temporal and parietal regions

was also evidenced by a [(99 m) Tc-HM-PAO] SPECT

study on our patient. The discordance between the neuro-

imaging data might refer to the different ages of the two

patients, to the presence of an underestimated cerebrovas-

cular disease, even though Guerreiro did not describe any

vascular risk factors affecting their case, or to a particular

phenotypic expression of this mutation occurring in one

case but not in the other. The PSEN2 R71W variant does

not affect Ab40/Ab42, as demonstrated by To et al. [10].

Thus, according to our results, a pathogenic mechanism

different from the classic one described for presenilins

mutations could be possibly hypothesized, involving other

still unidentified PSEN2-linked pathways. The presence of

R71W variant in a young subject with AD is strongly

suggestive of its pathogenic role in determining the disease

as no other mutations in Alzheimer’s causative genes were

found in our subject. In conclusion, this paper contributes

to revealing the clinical manifestations of this mutation

characterized by deficit in executive functions and behav-

ioural disturbances in an early onset case. Moreover, our

findings suggest that further in depth study is needed of the

allelic variants, which do not affect Ab40/Ab42 ratio in

AD cases with atypical phenotypes.

Conflict of interest None.

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