Novel mutations in the amyloid precursor protein gene within Moroccan patients with Alzheimer's disease

Novel Mutations in the Amyloid Precursor Protein Gene within Moroccan Patients with Alzheimer's Disease

Nadia El Kadmiri_&Nabil Zaid_&Ahmed Hachem_&

Younes Zaid_&Marie-Pierre Dubé_&Khalil Hamzi_&

Bouchra El Moutawakil&Ilham Slassi&Sellama Nadifi

Received: 23 December 2013 / Accepted: 27 February 2014

#Springer Science+Business Media New York 2014

Abstract

In Morocco, Alzheimer's disease (AD) affects almost 30,000 individuals, and this number could increase to 75,000 by 2020. To our knowledge, the genes predisposing individuals to AD and predicting disease incidence remain elusive. In this study, we aimed to evaluate the genetic con- tribution of mutations in the amyloid precursor protein (APP) gene exons 16 and 17 to familial and sporadic AD cases.

Seventeen sporadic cases and eight family cases were seen at the memory clinic of the University of Casablanca Neurology Department. These patients underwent standard somatic neurological examination, cognitive function assess- ment, brain imaging, and laboratory tests. Direct sequencing of exons 16 and 17 of the APP gene was performed on genomic DNA of AD patients. In this original Moroccan study, we identified seven novel frameshift mutations in exons 16 and 17 of the APP gene. Interestingly, only one novel splice mutation was detected in a family case. There is a strong correlation between clinical symptoms and genetic factors in Moroccan patients with a family history of AD. Therefore, mutations in APP gene exons 16 and 17 may eventually become genetic markers for AD predisposition.

Keywords

Moroccan patients . Alzheimer's disease . Frameshift mutations . APP gene

Introduction

Alzheimer's disease (AD) is the most common cause of de- mentia in the elderly, accounting for 50–56 % of cases, as assessed by autopsy and clinical series. Furthermore, it has an annual incidence of approximately 3 % in the 65–74 age group [Castellani et al.

2010]. This incidence rate doubles

with every increment of 5 years above the age of 65, as 1,275 new cases are diagnosed per year per 100,000 individ- uals older than 65. In Morocco, AD affects almost 30,000 individuals, and this number could increase to 75,000 by 2020 (projections of the World Health Organization (WHO)).

However, this disease remains poorly understood by the Moroccan general public. To our knowledge, the genes pre- disposing individuals to AD and predicting disease incidence remain elusive.

AD is clinically characterized by progressive memory im- pairment and deficits in cognitive functions. To date, there is no specific marker of AD [Bertram et al.

2010]. The diagnosis

is mainly clinical and is based on a set of revealing dysfunc- tion criteria based on neurological, cognitive, and behavioral assessments, as well as exclusion of other causes of dementia.

A definitive diagnosis of AD can only be made by autopsy, and several inclusion criteria are well established, such as the DSM IV-TR [DSM IV-TR

2003].

AD is pathologically characterized by the extracellular deposition of

β

-amyloid (A

β

) in senile plaques and the for- mation of intracellular neurofibrillary tangles, mainly com- posed of the hyperphosphorylated microtubule-associated protein tau [Gomez-Ramos et al.

2004]. To date, three genes

have been identified as highly penetrant but rare mutations causing early-onset familial AD (EOAD) and showing nearly

N. El Kadmiri (*)

:

K. Hamzi

:

B. El Moutawakil

:

I. Slassi

:

S. Nadifi

Laboratory of Medical Genetics and Molecular Pathology, Faculty of Medicine and Pharmacy, University Hassan II, 19 Rue Tarik Ibnou Ziad, B.P. 9154,

20000 Casablanca, Morocco e-mail: elkadmiri1979@gmail.com B. El Moutawakil

:

I. Slassi

Department of Neurology, CHU IBN ROCHD, Casablanca, Morocco

N. Zaid

:

A. Hachem

:

Y. Zaid

:

M.<P. Dubé Montreal Heart Institute, Montreal, Canada DOI 10.1007/s12031-014-0278-7

100 % penetrance with autosomal dominant inheritance.

These genes are the amyloid precursor protein (APP) gene, which code for APP, presenilin-1 gene (PS1), and presenilin-2 gene (PS2), respectively [Schellenberg

1995]. With respect to

the more common, late-onset form of AD, a polymorphism in the apolipoprotein E gene (ApoE

ε

4) has been established as a major risk factor for complex forms of AD, mainly in sporadic late-onset AD (LOAD) cases [Fei and Jianhua

2013]. More

recently, genetic studies of AD have focused on identifying common variants associated with risk of LOAD through genome-wide association studies. These studies have identi- fied several new genes that show significant association with LOAD, including CLU, BIN1, PICALM, and ABCA7, each with only a small effect [Harold et al.

2009].

Cell-based studies and mouse models have shown that muta- tions in genes encoding APP, PS1, and PS2 cause an increased production of the neurotoxin amyloid-β 42 (Aβ42) [Theuns and Van Broeckhoven

2000], indicating that unbalanced APP pro-

cessing may be the primary event leading to the neurodegener- ative brain pathology in AD patients carrying these mutations. At present, more than 130 mutations have been described in PS1 gene, only 8 in PS2 gene, and about 20 in the APP gene (available at:

http://molgen-www.uia.ac.be/ADmutations).

Some mutations in the APP gene are pathogenic, while others are silent mutations or polymorphisms [Murrell et al.

1991].

Methods

Patient Recruitment

Patients were followed since 2004 by the Memory Consultation Group at the CHU IBN ROCHD Neurology Department in Casablanca, Morocco. The protocol was ap- proved by the human ethical committee of the CHU IBN ROCHD in accordance with the declaration of Helsinki for experiments involving humans, and written consent was ob- tained from the patients and their guardians prior to the study.

Seventeen sporadic cases and eight family cases were seen at the memory clinic of the Neurology Department of the University of Casablanca Hospital IBN ROCHD. A family history was obtained by a clinical interview of the patient and a

“yes or“no self-reporting questionnaire from the guardian

or other family member. The disease was considered familial if at least one additional first-degree relative suffered from EOAD-type dementia. All patients underwent standard somat- ic neurological examination, cognitive function assessment, brain imaging, and laboratory tests (Table

1).

Assessment of cognitive function varied according to the education level of the subject, and it included at least one mini-mental state examination (MMSE), as recommended by the health high authority. The examination was quoted on 30 points, including investigation of orientation, learning,

attention, calculation, immediate memory, language, and the ability to execute simple orders. It is not a specific diagnostic test for AD, but it puts into evidence the severity level of AD as well as the deterioration of cognitive function [Folstein et al.

1975]. The deterioration stages are termed as follows:

“light for scores between 20 and 26,“moderate for scores

between 15 and 19,

“moderate severe for scores between 10

and 14, and

“

severe for scores below 10. However, there is no consensus regarding these limits, and a score is attributed for each patient. Several elements may modify the assessment test results, such as the patient's age and sociocultural status, which are important factors to take into consideration when interpreting the test results. In our cases, in addition to the MMSE, according to the level of education, the clinician uses other tests that do not require necessarily a level of education such as BEC96 [Signoret et al.

1988], visual short-term or

digital memory assessment, work memory assessment, lan- guage assessment test (DO80) [Deloche and Hannequin

1997], and apraxia.

Brain magnetic resonance imaging (MRI) was routinely performed on all patients; however, due to financial limita- tions, a brain scan alone was performed on a number of subjects. No patient underwent functional imaging. The bio- logical assessment consisted mainly of blood analysis for complete blood count, liver, renal, and thyroid function, as well as vitamin B12 and B9 serum levels. However, depend- ing on the clinical context, other tests were performed.

Genomic Studies

Genomic DNA Extraction and Amplification of the APP Gene

Genomic DNA was isolated from peripheral blood leukocytes using the salting out procedure. In this conventional tech- nique, proteins and other contaminants are precipitated from the cell lysate using high concentrations of salt such as potas- sium acetate or ammonium acetate. The precipitates are re- moved by centrifugation, and the DNA is recovered by alco- hol precipitation. The genomic DNA samples were stored at

−20 °C. For each patient, polymerase chain reaction (PCR)

was performed for APP gene exons 16 and 17.

Genomic DNA Sequencing and Analysis

Direct sequencing of the APP gene exons 16 and 17 was performed on the genomic DNA of AD patients and healthy

Table 1 Subject distri-

bution according to gen- der and AD type

Mmale,Ffemale

M F Total

Family cases 4 4 8

Sporadic cases 7 10 17

Total 11 14 25

controls. Briefly, Sephadex purified PCR products were se- quenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) with the same forward and reverse primers. Since the 1,000 genome database allows for the detection of most genetic variants that have frequencies of at least 1 % in the populations studied, we chose this database as a study tool. ANNOVAR, PYTHON, and BLAT UCSC pro- grams were used to evaluate these mutations.

Results

In our study of 25 individuals, the observed mean age of AD patients was 64.52±9.30, and we found a slight female pre- dominance (56 vs 44 %). In addition, we found a prevalence of AD of approximately 20 %, which increases with age, in the population below 60 years of age. Approximately half of our patients (48 %) have a score lower than 10 and are affected by severe dementia, while 28 % are affected by moderate severe dementia and 12 % are lightly to moderately insane.

Twenty-five patients underwent neuroimaging, 18 of whom were assessed by MRI, while 7 were assessed by computed tomography (CT). All patients had hippocampal and parahippocampal atrophy. The blood work showed no abnor- malities in the 25 enrolled AD patients.

Sequence analysis of the APP gene exons 16 and 17 revealed seven novel frameshift mutations in 17 sporadic cases and 8 family cases. Although no mutations in exon 16 of the APP gene were found in the 17 evaluated sporadic cases (Table

2), no mutations in exons 16 and 17 of the APP gene

were found in healthy controls.

The first novel mutation 27269957-G at codon 589, which is caused by a single-nucleotide insertion, was identified in three family cases. The first family case harbors a female patient (ID P20) with a disease onset age of 60 and a positive family history of EOAD (Fig.

1

(pedigree 1)). Her MMSE score was 24/30, and her first experienced symptom was progressive impairment of memory. Analysis of her MRI revealed a hippocampal and cortical atrophy (Fig.

2a, b). In

this patient, only one intronic substitution (27270075A>G) was detected in the analyzed regions of the APP gene at exon 16.

The second family case harbors a female patient (ID P31) with an onset age of 63 and a positive family history of EOAD (Fig.

1

(pedigree 2)). Neuropsychological examination at the age of 68 showed severe cognitive impairment. The early progressive impairment of episodic memory was noticed 5 years ago. The patient presented with progressive memory and language impairment, aphasia, visuospatial disorientation, decreased autonomy, executive dysfunction, and praxis defi- cits, all leading to severe dementia. Neuroimaging revealed hippocampal and cortical atrophy (Fig.

2c, d). The MMSE

score was not evaluated as the patient is illiterate, but the

Table2FrameshiftmutationsidentifiedinEOADandLOADAPPgeneexons16and17inMoroccancaseswithprobableAD GenePatientIDExonGenomicmutationMutationtypeCodingproteinDescribedinSexOnsetage/durationFamilyhistoryFirstclinicalMMSEscoreMRI/CT nameANNOVARofillness(years)ofdementiasymptom(s) APPP20162127269957-GFrameshiftc.1767_1768insCNoF60/7YESMemoryloss24/30Atrophy P31162127269957-GFrameshiftc.1767_1768insCNoF63/5YESMemorylossIlliterateAtrophy P32162127269957-GFrameshiftc.1767_1768insCNoF64/2YESMemorylossIlliterateAtrophy P30162127269960-GFrameshiftc.1764_1765insCNoM63/2YESMemoryloss18/30Atrophy P2016Intronic27270075A>G–NoncodingNoF60/7YESMemoryloss24/30Atrophy P24172127264134-GFrameshiftc.1886_1887insCNoM49/2YESMemoryloss30/30Atrophy P24172127264052A-Frameshiftc.1968delTNoM49/2YESMemoryloss30/30Atrophy P33172127264139-CFrameshiftc.1881_1882insGNoM60/2NOMemoryloss10/30Atrophy P2717SplicingAPPFrameshiftc.1932+2T>-NoF59YESNo30/30N/A P1817Intronic27264009-T–NoncodingNoM55/6YESMemoryloss5/30Atrophy P3172127264134-GFrameshiftc.1886_1887insCNoM56/2NOMemoryloss15/30Atrophy P417Intronic27264240T-–NoncodingNoM51/8NOMemorylossN/AAtrophy MMSEmini-mentalstateexamination,Ffemale,Mmale

clinician uses other tests that do not require necessarily a level of education such as BEC96, visual short-term or digital memory assessment, work memory assessment, language as- sessment test (DO80), and apraxia. The patient has one living sister who likely has AD, as well as one sister who likely died of AD. However, no postmortem examination was performed on the deceased.

The third family case harbors a female patient (ID P32) with a disease onset age of 64. The first symptom presented by the patient was progressive memory impairment, which was observed during a 2-year period. The patient's MRI images showed a hippocampal and cortical atrophy. In addition, the patient's mother is probably afflicted with AD (Fig.

1

(pedigree 3)).

The second novel 27269960-G mutation at codon 588, which is caused by a single-nucleotide insertion, was found in one male case (ID P30) with a family history of AD (Fig.

1

(pedigree 4)). The patient's disease onset age was 63, and his symptoms of progressive dementia had developed 2 years prior to AD diagnosis. The patient's MMSE score was 18/

30, and his neuropsychological examination revealed cogni- tive impairment, specifically memory deterioration. In

addition to the patient, four of his family members, namely one sister, two brothers (one is deceased), and his mother (deceased), were likely afflicted with AD.

In our patient population, four novel frameshift mutations were identified in exon 17 of the APP gene, including one novel splice mutation and two intronic mutations, as described below.

The first two novel frameshit mutations 27264134-G and 27264052-A that are caused by a single-nucleotide insertion at codon 629 and a single-nucleotide deletion at codon 656, respectively, were identified in a male patient (ID P24) with a disease onset age of 49 and a family history of AD (Fig.

1

(pedigree 5)). The patient's MRI images revealed a hippocam- pal atrophy while his neuropsychological examination at the age of 50 showed memory impairment, aphasia, and praxis deficits as well as an MMSE score of 30/30. In addition, the patient's mother, 75 years of age, was likely afflicted with AD since she showed signs of generalized severe cognitive impair- ment. Her disease onset age was 66, which was followed by severe dementia 9 years later. Both of the patients' sisters agreed to participate in the study. However, only one sister (ID P27), aged 59, carries the splice mutation c.1932+2 T>-, which is

Fig. 1 Pedigrees representing

families with cases in which APP exons 16 and 17 frameshift mutations were observed.Roman numbersto the left of the pedigrees denote generations.

Numbersbelow the patient symbols denote order of patients.

Arrowsindicate the probands

caused by a single-nucleotide deletion. Her neuropsychological tests showed no cognitive impairment, and her MMSE score was 30/30.

The third novel frameshift mutation 27264139-C, which is caused by a single-nucleotide insertion at codon 627, was identified in a 62-year-old male patient (ID P33). The patient's initial symptoms were observed at the age of 60, which were progressively followed by memory deficits 2 years later. In addition to the patient, his deceased mother was likely afflicted with AD. Neuroimaging revealed hippocampal and cortical atrophy (Fig.

2e, f). The MMSE score was 10/30.

The two intronic mutations were identified individually.

The first mutation was detected in a family case in a male patient (ID P18) with a disease onset age of 55 and a family history of AD. After a 6-year observation period, the patient had developed severe cognitive impairment, specifically memory and praxis deficits. The patient's MRI images re- vealed a hippocampal and cortical atrophy. His MMSE score was 5/30. The second intronic mutation was detected in a sporadic case in a male patient (ID P4) with no family history

of AD. The patient's disease onset age was 51, and his MRI images revealed diffuse cortical atrophy. The patient had developed generalized severe cognitive impairment 8 years following disease onset.

The last frameshift mutation 27264134-G, which is caused by a single-nucleotide insertion, was detected in a sporadic male case (ID P3) with no apparent family history of AD. The patient's MRI images revealed diffuse cortical atrophy where- as his neuropsychological examination showed memory and praxis deficits and an MMSE score of 15/30.

Discussion

AD is caused by the accumulation of amyloid plaque buildups in the brain. These plaques are partially composed of Aβ, which is a fragment derived from APP. Therefore, a mutation in the APP gene is believed to account for 5 to 20 % of all EOAD, and individuals harboring such mutations develop AD at the approximate age of 50 [Mayeux

2010]. To date,

several pathogenic mutations have been found in exons 16 and 17 of the APP gene (available at:

http://molgen-www.uia.

ac.be/ADmutations). However, these are missense mutations,

and according to the APP770 numbering system, the mutations associated with EOAD are APP715, APP716, APP717, and APP670/671 that correspond to Val715Met, Ile716Val, Val717Ile/Gly/Phe, and Lys670Asn/Met671Leu, respectively. These missense mutations are positioned in a region outside the Aβ sequence [Goate et al.

1991].

In this original study, we identified seven novel frameshift mutations in exons 16 and 17 of the APP gene, of which five were identified in familial AD cases and two in sporadic AD cases. Interestingly, only one novel splice mutation was de- tected in a family case. These frameshift mutations are caused by either an insertion or a deletion of a single nucleotide in the gene, which changes the reading frame due to a codon shift.

An insertion or deletion early in the sequence of a gene results in a more altered protein, which could be abnormally short or long and most likely nonfunctional [Van Den Hurk et al.

2001]. Generally, mutations in the APP gene account for

approximately 5 % of familial AD, with a disease onset age of 65 [Rocchi et al.

2003]. In our study, we show that the

frequency of frameshift mutations in exons 16 and 17 of the APP gene is 87 % (7 out 8 cases) within familial AD cases, whereas the frequency of mutations in exon 17 is 12 % (2 out of 17 cases) within sporadic AD cases.

In exon 16 of the APP gene, two novel frameshift muta- tions were detected in four family cases. The first mutation that occurs at codon 589 was identified in three family cases (ID P20, ID P31, and ID P32) and is caused by a single- nucleotide insertion. In these three cases, the age of onset of the disease varied between 60 and 64, and there was a family history of AD. The second mutation that occurs at codon 588

Fig. 2 MRI brain.ID P20axial cut (a) and sagittal cut (b). ID P31

sagittal cut (c) and coronal cut (d).ID P33sagittal cut (e) and coronal cut (f) revealing hippocampal and cortical atrophy

was identified in one male patient (ID P30) with a family history of AD and an age of onset of 63.

In our cases, four novel frameshift mutations were identified in exon 17 of the APP gene, including one splice mutation and two intronic mutations. The two frameshift mutations that are caused by a single-nucleotide insertion at codon 629 and a single-nucleotide deletion at codon 656 were identified in a male patient (ID P24) with a family history of AD and an age of onset of 49. Interestingly, the splice mutation, which is caused by a single-nucleotide deletion, was identified in this patient's 59-year-old sister (ID P27). The third frameshift mutation, which is caused by a single-nucleotide insertion at codon 627, was identified in a 62-year-old male patient (ID P33).

While this case is considered sporadic, the patient's mother was likely afflicted with AD at the time of her death. Therefore, this is not exclusively a sporadic case. The fourth frameshift muta- tion, which is caused by a single-nucleotide insertion, was detected in a sporadic case in a male patient (ID P3) without a complete evidence of a lack of family history of AD.

Neuropsychological evaluation is an important step in AD diagnosis, and it is based on several standardized psychomet- ric tests such as the MMSE [Folstein et al.

1975], the Blessed

Scale, the Global Deterioration Scale (GDS), and the Alzheimer Disease Assessment Scale-cognitive subscale (ADAS-cog) [Reisberg et al.

1982; Rosen et al. 1984].

However, the MMSE remains the most utilized test because it allows for a quick evaluation of cognitive functions and is required for the diagnosis of insanity, according to the NINCDS-ADRDA criteria.

In our cases, according to the degree of severity of the disease, all individuals had hippocampal atrophy and cortical atrophy accompanied by ventricular extension, which was prevalent in 62 % of the familial cases and 47 % of the sporadic ones. At early stage, patients had hippocampal atro- phy that leads to memory disorders, which progresses with the disease to affect other brain regions. Indeed, the hippocampal volume was significantly reduced in patients afflicted with AD as compared with normal subjects. However, despite the lack of accurate quantification of the level of atrophy in our cases and according to other studies [Grundman et al.

2002;

Korf et al.

2004], we could conclude that a higher level of

atrophy reflects a decrease in neuropsychological perfor- mances. The cholinesterase inhibitors improve cognitive out- comes in such AD patients, but the benefits of these drugs for behavioral disturbances are unclear [Greenblatt et al.

2003;

Howard et al.

2007]. All patients received cholinesterase

inhibitors associated to antidepressants according to the state of disease severity.

The clinical, neuropathological, and genetic assessments of mutated APP-linked familial AD in our Moroccan cases have several shared features. For instance, there is a strong correla- tion between clinical symptoms and genetic factors in our Moroccan cases with a family history of AD. Moreover, all

evaluated frameshift mutations were associated with clinical symptoms of AD, which may explain in part the reason behind the disease in these patients and the disease inheritance throughout the generations. It is worth noting that frameshift mutations are more harmful than base substitution mutations because the outcome of a frameshift mutation is a complete alteration of the amino acid sequence of the protein. This alteration is due to a shift in the reading frame of the transcribed messenger RNA (mRNA), which starts at the codon where the mutation occurs. The resulting protein is completely altered or nonfunctional following mRNA translation by ribosomes. In contrast, a three-nucleotide insertion or deletion within the gene does not shift the reading frame but rather inserts an extra amino acid or removes one from the final protein.

The discovery that mutations in genes that encode APP, PS1, and PS2 are linked to familial AD has ushered in a new and exciting era of research aimed at clarifying the relation- ship between genetic abnormalities and AD pathogenesis.

However, the genes associated with autosomal dominant in- heritance of familial AD remain to be identified.

Acknowledgments We would like to thank Dr. F.D. Tiziano and all of the staff at the Universita Catholica in Rome, Italy, for their help in gene sequencing. We thank Ranbanxy Morocco LCC for their partial financial support to Miss Nadia El Kadmiri. We thank Mr. Othman Rouissi for his help. We appreciate the assistance offered by the staff at the CHU IBN ROCHD Neurology Department in Casablanca and the staff at the Lab- oratory of Medical Genetics and Molecular Pathology, FMPC, specifi- cally Pr Hind Dehbi and Mr. Said Wifaq.

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