Novel Mutations in the Amyloid Precursor Protein Gene within

J Mol Neurosci DOI 10.1007/s12031-014-0278-7

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 contribution 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 assessment, 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.

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: [email protected] B. El Moutawakil : I. Slassi Department of Neurology, CHU IBN ROCHD, Casablanca, Morocco N. Zaid : A. Hachem : Y. Zaid : M.-, which is

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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 revealed 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

Fig. 2 MRI brain. ID P20 axial cut (a) and sagittal cut (b). ID P31 sagittal cut (c) and coronal cut (d). ID P33 sagittal cut (e) and coronal cut (f) revealing hippocampal and cortical atrophy

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 whereas 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 detected 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 mutations 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 singlenucleotide 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

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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 mutation, 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 psychometric 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 atrophy 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 performances. The cholinesterase inhibitors improve cognitive outcomes 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 correlation 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 relationship between genetic abnormalities and AD pathogenesis. However, the genes associated with autosomal dominant inheritance 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 Laboratory of Medical Genetics and Molecular Pathology, FMPC, specifically Pr Hind Dehbi and Mr. Said Wifaq.

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