Probable novel PSEN2 Val214Leu mutation in Alzheimer’s disease supported by structural prediction

Well-known mutations associated with autosomal dominant Alzheimer’s disease (AD) inheritance are amyloid precursor protein (APP) and presenilin 1 (PSEN1) and presenilin 2 (PSEN2) [1, 2]. Previous studies have shown several APP and PSEN1 mutations in Asian populations, but pathogenic PSEN2 mutation has not yet been described [3, 4]. We had screened PSEN2 mutation in 90 AD patients of two memory clinics from May to December 2012. Here, a patient of East Asian descent with AD was found with a probable novel Val214Leu mutation at PSEN2 exon 7.

A seventy-year-old right-handed female has complained progressive memory problems, which started one year prior to the initial visit. She reported her frequent forgetfulness of going to important meetings and had difficulties in grocery shopping. She also reported in having a tremor in her right hand for the past 20 years, persistent during resting and forearm stretching stances, which did not progress further. She presented with slightly decreased facial expressions, but did not show rigidity, ataxia, myoclonus, or seizure. Since she was separated from her other family members during the Korean War, we could not obtain her family history. Her Korean version of Mini-Mental Status Examination (K-MMSE) was 18 and CDR-sum of boxes was 4.5. She also underwent neuropsychological tests using a standardized neuropsychological battery called the Seoul Neuropsychological Screening Battery (SNSB) [5]. She had normal spontaneous speech and comprehension. Her SNSB showed digit span forward was 16.86%ile; Korean-Boston naming test, 12.09%ile; Rey-Osterrieth Complex Figure Test copy, 0.01%ile; calculation, < 5%ile; Seoul Verbal Learning Test-delayed recall, 1.05%ile; Rey-Osterrieth Complex Figure-delayed recall test, 2.09%ile; Digit Symbol Coding, 1.15%ile; Korean Stroop test-color reading, 11.46%ile; Controlled Oral Word Association Test, 0.01%ile. She also revealed the BPO (body part as object) error when doing praxis test. Fist-edge-palm and alternating hand movement, motor impersistence test were normal, but Luria loop and alternating square and triangle were deformed and perseverative. Neuropsychological tests revealed severe verbal memory impairment, which was not improved by cues. In addition, she showed visuo-spatial dysfunction and difficulty in calculation and frontal executive function. Her brain MRI (Figure 1a) showed diffuse cortical atrophy with mild white matter hyperintensities in FLAIR images, and FDG-PET (Figure 1b) revealed bilateral temporoparietal and precuneus hypometabolism. A diagnosis of probable AD was made. She had an APOE ϵ3/4 polymorphism, but other laboratory tests were normal.

DNA was extracted from the buffy coat of the venous blood samples using a blood DNA isolation kit (GeneAll Inc., Seoul, South Korea). APP exon 16 and 17, PSEN 1 exon 4, 5, 6, 7, 8, and 11, PSEN 2 exon 4, 5, 6, 7, and 12, the coding region of the PRNP gene, mutations in the microtubule-associated protein tau (MAPT) and progranulin (GRN) were amplified by PCR for genetic analysis. A sizing-genotyping and repeat-primed PCR were performed to monitor the presence of the abnormal repeat expansions of C9orf72.

Single-strand conformation polymorphism (SSCP) [6] and Surveyor nuclease assay kit [7] revealed alternative migration and cleavage patterns in comparison to normal (Figure 2a, b), strongly suggesting the presence of mutation in the PCR product of PSEN 2 exon 7. All PCR products were sequenced for the confirmation. We found a valine to leucine substitution (Val214Leu) at exon 7 of PSEN2 in both patients (Figure 2c). This PSEN2 mutation could be considered as a novel mutation, since 614 control chromosomes (307 subjects: 140 healthy control, 90 AD, 15 mild cognitive impairment, 12 subjective memory impairment, 10 frontotemporal dementia, 5 vascular cognitive impairment, 35 unclassified patients) were also screened for Val214Leu mutation without any evidence, in support of the present finding. All study subjects provided written informed consent to allow their genetic and clinical data to be used for research purposes. The Institutional Review Board at Chung-Ang University Hospital and Seoul National University Bundang Hospital approved this study.

The structures of presenilin 2 with native Val 214 residue and Leu 214 mutation were generated by RaptorX 3D prediction program. Overall structural features were identical between the native presenilin 2 with Val 214 residue and Leu mutation. However, the side-chains of Val and Leu 214 residues reveal noticeable changes of extending towards opposite direction. In addition, other proceeding residues, Ile 219 and His220, revealed significant changes in their side-chains (Figure 3).

Polymorphism Phenotyping v2 (PolyPhen 2, http://​genetics.​bwh.​harvard.​edu/​pph2/​) online program for predicting the putative pathogenic phenotype was also used to evaluate PSEN2 Val214Leu mutation by analyzing the non-synonymous mutations in the coding region [8]. PolyPhen2 compared the wild-type sequences and the mutant sequences by using sequence-and structure-based predictive features, based on maping the missense mutations to the transcriptomes, performes protein sequence annotations and structural comparisons. Next, PolyPhen 2 performed a multiple alignment of sequences from different organisms. In addition, it could provide 3D structure of the protein by searching protein data bank database. Two types of datasets, HumDiv and HumVar, were available from PolyPhen 2, which were calculated from position-specific independent counts (PSIC) scores of normal and mutant allele. For PSEN2 Val214Leu, the HumDiv score was 0.972 (sensitiviy: 0.77; specificity: 0.96), which indicated as probably damaging mutation with the HumVar score of 0.836 (sensitiviy: 0.73; specificity: 0.88), as possibly damaging mutation.

We detected a single nucleotide polymorphism on exon 7 of PSEN2 in a patient with dementia of the Alzheimer type. So far, a pathological PSEN2 mutation has not been reported in Asia. Particularly, Val214Leu at PSEN2 was not reported previously and also was not found in general Korean population. This is suggesting as a probable novel PSEN2 mutation in association with this AD patient. Upon structural prediction of native and mutant presenilin 2 by using RaptorX program, the results revealed significant structural changes in the region (Figure 3). When two structures of Val 214 and Leu mutant were superimposed, the where side-chains of Val 214 and Leu 214 were pointing to opposite direction, suggesting drastic alterations in the region. Additional residues, Ile219 and His220, also revealed dramatic changes, supporting that Val replacement to Leu could cause overall structures and function of presenilin 2 (unpublished result, An’s personal communication). On the other hand, many other pathogenic mutations of Val replacement to Leu were reported [9, 10].

PolyPhen2 and other bioinformatic algorithms could be used for predicting the pathogenic nature of novel mutations or known mutations [11, 12]. Since cell models and in vitro/in vivo experiments would be costy, in silico estimations and simulations could aid in understanding the pathogenic or non-pathogenic nature of mutations. The prediction for PSEN2 Val214Leu by Polyphen2 provided HumDiv and HumVars scores of 0.972 and 0.836. Hence, These analyses suggested the PSEN2 Val214Leu mutation could be a possibly strong damaging variant.

Multiple sequence alignment for PSEN2 was performed in comparison with different organisms. Analysis predicted that the vertebrates, such as rhesus macaque, african elephants pigs, dog, duck, revealed the normally codon Val at 214. However, few organisms (invertebrates, mostly unicellular organisms) revealed the alterations, Leu, Gly, Ile or Met, at the same Val214 position. For example, Methionine, isoleucene, or leucine were found in the Tribolium castaneum, Trichoplax adhaerens, and Entamoeba histolytica, respectively. PSEN2 V214 seemed to be strongly conserved in 115 species and mutations of Leu, Gly, Ile or Met were found in 8 species.

AD patients who have exon-7 PSEN2 have reported wide spectrum of cognitive and non-cognitive symptoms. They could initially show anxiety and irritability along with mild deficits in memory, attention, and language. Then they could also show seizure and myoclonus. Some patients present global cognitive impairment at first. Exon-7 PSEN2 mutation revealed an age range of onset between 45 and 85 years overlapping early and late onset AD [13‐15]. This could be explained by the demonstrated tendencies of PSEN2 mutations in presenting lower penetrance and appearing a later and more variable age of onset than the other early-onset AD gene mutations [16]. Although the patient showed a multi-domain cognitive impairment with decreased facial expression and non-progressing right hand tremor, her significant memory impairment was the initial main complaint at age 69 and prominent temporoparietal lobe dysfunction supported by FDG-PET, no abnormal repeat expansion of C9orf72 and no other mutation in MAPT and GRN, which could be clinically classified as late-onset AD. Because her family was separated during Korean War, family history was limited. We could not find any other affected family member.

When considering diagnoses atypical AD patients, we can do CSF Aβ 1-42 analysis or Pib-PET supporting an AD diagnosis. Even though she did not perform CSF and Pib-PET study, we considered the patient as probable AD because she initially complained memory problem confirmed with a neuropsychological test and showed bilateral hippocampal atrophies in structural MRI and hypometabolism of bilateral temporoparietal area and precuneus in FDG-PET.

This is a case report. This manuscript has not been published elsewhere, nor is it under simultaneous consideration for publication elsewhere. All of the authors and contributors have agreed to the conditions of authorship. All authors have full access to all of the data and have the right to publish any and all data, and each author believes that the manuscript represents honest work. The methods section includes a statement of our institutional review board (IRB)’s approval. This research complies with the principles of the Declaration of Helsinki (1964). No identifying information for the patient is included in the data reported. There are no conflicts of interest to report and all authors adhered to ethical standards.