The novel I213S mutation in PSEN1 gene is located in a hotspot codon associated with familial early-onset Alzheimer's disease

Mutations in presenilin 1 gene (PSEN1) are the most common causes of autosomal dominant early-onset Alzheimer's disease (EOAD). We report a novel PSEN1 mutation (I213S) that was discovered in an Italian patient with a family history of early-onset dementia, who developed a slowly progressive cognitive decline since the age of 40 years. Clinical investigations, including neuropsychological assessment, brain MRI and 18-fluorodeoxyglucose PET, as well as cerebrospinal fluid biomarkers, supported the diagnosis of EOAD. Genetic studies identified a novel missense mutation at codon 213 (I213S). Three other mutations at the same codon have been described in association with EOAD. Previous in silico, in vitro and in vivo studies indicated that these mutations affect the functional properties of γ-secretase and are most likely pathogenic. In silico algorithms suggested that even the I213S mutation has similar deleterious effects on PSEN1 structure and function. Overall, these data strongly support a role of hotspot site for the codon 213 of PSEN1, and provide evidence that the genetic variants located on this site cause EOAD.


Introduction
Alzheimer's disease (AD) is the leading cause of dementia in the elderly ( Jack et al., 2019 ;Reitz et al., 2011 ). About 95% of AD cases occur sporadically and start late in life (late-onset AD, LOAD). There is considerable evidence supporting a strong genetic component in its etiology ( Farrer et al., 1990 ), as LOAD is likely caused by multiple low penetrance genetic variants ( Naj and Schellenberg, 2017 ). In about 5% of AD patients the disease has an early onset, before 65 years of age, and is most often familial (early-onset AD, EOAD) ( Chouraki and Seshadri, 2014 ). EOAD is characterized by highly penetrant mutations in a few known risk genes: the majority of cases are in fact linked to mutations in 3 causative genes encoding amyloid precursor protein ( APP ), presenilin 1 ( PSEN1 ), and presenilin 2 ( PSEN2 ). Most of EOAD cases are associated with positive family history of dementia, but sev- Mutations in PSEN1 impair the γ -secretase functions in APP cleavage, resulting in elevated A β42 and/or reduced A β40 production ( Xia et al., 2015 ). According to the amyloid cascade hypothesis, the prevalence of long A β42 isoform is thought to promote the formation of pathogenic assemblies of A β which trigger the series of molecular events leading to the disease ( Hardy and Higgins, 1992 ). Marked heterogeneity of familial AD phenotypes reported for various PSEN1 mutations at the clinical and neuropathological levels suggests that AD pathogenesis linked to PSEN1 mutations is more complex and can involve additional cellular mechanisms ( Bialopiotrowicz et al., 2012 ).
In the present study we report a novel PSEN1 mutation, I213S, associated with familial EOAD, located in a mutational hot spot codon of the gene and probably impairing the functional properties of γ -secretase. Our findings support the pathogenic nature of this genetic variant.

Subjects
Here we describe the clinical picture of a young woman and her family members with early onset dementia. A 3-generation family tree was build based on available information obtained from hospital records and anamnestic interview of relatives. This study was approved by the local research ethics committee. Written informed consent was obtained from participants.

DNA purification and genetic testing
Genomic DNA was extracted from peripheral blood lymphocytes using standard protocols. All the coding exons of PSEN1 and PSEN2 genes and exons 16-17 of APP gene was amplified by PCR using specific primers, bi-directionally sequenced using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on a 3130xl Genetic Analyzer System (Applied Biosystems).

In silico analyses
The evolutionary conservation of the 213 Isoleucine among species was assessed by the ConSurf web server. The functional effects and the pathogenicity of the mutation were predicted us-

Clinical findings
The proband ( Fig. 1 A, subject III-2) was a 42-year-old female patient having a remarkable family history of dementia in the maternal lineage. Indeed, an early-onset progressive cognitive decline was reported in her older sister ( Fig. 1 A, III-1), her mother  Fig. 1 A,II-2), and her grandmother ( Fig. 1 A, I-1). Her grandmother was affected by an early-onset cognitive decline and died at the age of 35. Proband's mother was affected by progressive cognitive deterioration since the age of 40 years and died at the age of 56. Proband's sister developed a slowly-progressive cognitive decline since the age of 30 years, associated with mood depression and anxiety, non-responsive to treatment with antidepressant drugs. Serial neuropsychological evaluations documented an amnesic syndrome initially diagnosed as amnesic single-domain Mild Cognitive Impairment (MCI). Brain MRI and 18 F-Fluorodeoxyglucose positron emission tomography and/or computed tomography ( 18 F-FDG PET/CT), performed at the beginning of her neurologic illness, were unremarkable. However, the study of neurodegenerative biomarkers in cerebrospinal fluid (CSF) showed increased levels of total tau (t-tau: 1200 pg/mL, normal range < 300 pg/mL) and phosphorylated tau (p-tau: 170 pg/mL, normal range < 61 pg/mL), and low levels of the 1-42 fragment of A β peptide (A β42: 381 pg/mL, normal range > 500 pg/mL). Her cognitive deficits gradually evolved toward a full dementia with involvement of all main cognitive domains. Treatment with Donepezil, Rivastigmine and Memantine in different disease phases had only poor and transiently positive effects on her progressive cognitive decline, and she died at the age of 42 years Unfortunately, no genetic testing was carried out in the proband's affected family members, with the exception of ApoE genotype in her sister, who resulted to be carrier of the ɛ 3/ ɛ 3 polymorphism.
The proband's past personal medical history was unremarkable with the exception of a mild traumatic head injury with transient mental confusion in the following few days, 5 years before the onset of her cognitive decline, and the presence of slight psoriatic arthritis. She reported cognitive disturbances consisting of impairment of memory and mood dysregulation, characterized by fluctuant anxiety and depression, which began insidiously since the age of 40 years with a slow progression over the time. Treatment with SSRIs improved her emotion but hadn't substantial benefits on memory complain. At the age of 41, a first neuropsychological assessment documented the presence of memory deficits, and mild impairment in executive cognitive functions (MoCA test 19/30; FAB 15/18) with no substantial impact on daily living.
At the age of 42 she was admitted to our hospital because of increasing troubles in her job and housekeeping due to cognitive difficulties. Neuropsychological testing revealed a multi-domain MCI characterized by deficit in visuo-spatial and verbal anterograde episodic memory, working memory, and executive functions ( Table 1 ). The most relevant alterations in cognitive performances of the patient suggested a dominant impairment of the hippocampal network characterized by the rapid and complete amnesia for new information and even for the memory task presentation.
Brain MRI showed mild atrophy in frontal and parietal lobes of both emispheres ( Fig. 2 , panels A and B). 18 F-FDG PET/CT displayed severe hypometabolism at the level of posterior cingulus, precuneus and parietal lobe bilaterally ( Fig. 2 , panels C and D). A

Fig. 2.
Neuroimaging data for the proband carrying the PSEN1 I213S mutation. MRI showed slight and diffuse brain atrophy, particularly in the frontoparietal regions of the brain (panels A and B). 18-FDG PET scan showed bilateral hypometabolism of posterior cingulus, precuneus, and parietal lobe (panels C and D). Amy-PET documented a diffuse cortical tracer retention, with loss of contrast between white and gray matter, more pronounced in mesial frontal cortex, precuneus, superior gyri of lateral temporal cortex, and parietal lobes (panel E). Abbreviations: MRI, magnetic resonance imaging; PSEN1 , presenilin 1;18-FDG PET, Positron Emission Tomography using radiolabelled 18 fluorodeoxyglucose; Amy-PET, Positron Emission Tomography with radiolabelled β-amyloid tracer ( 18 F-Flutemetamol). Panels A, B, C: sagittal, coronal, axial MRI T1-weighted images, respectively; panel D: axial MRI FLAIR T2-weighted image. Unknown Unknown mild reduction of glucose uptake was also documented in bilateral prefrontal cortex and in the lateral occipital and lateral temporal areas with higher evidence in the right brain emisphere. Z-scores for the different brain areas analyzed by 18 F-FDG PET/CT are reported in supplementary fig. S1. PET scan with a β-amyloid ligand ( 18 F-Flutemetamol) revealed a diffusely increased uptake of the tracer in neocortex, particularly in mesial frontal cortex, precuneus, superior gyri of lateral temporal cortex and parietal lobes, indicating a clear accumulation of β-amyloid in the patient's brain ( Fig. 2 , panel E). Routine laboratory investigations in blood and urine samples were unremarkable as well as the standard cerebrospinal fluid (CSF) test. The study of neurodegenerative markers in CSF showed increased levels of total tau (t-tau: 1272 pg/mL, normal range < 466 pg/mL) and phosphorylated tau (p-tau: 317 pg/mL, normal range < 61 pg/ml), and low levels of the 1-42 fragment of A β peptide (A β42: 320 pg/mL, normal range > 550 pg mL). The CSF levels of the 1-40 fragment of A β (A β40) were 8316 pg/mL (normal range 7755-16715 pg/mL). The A β42/A β40 ratio was 0.03 pg/mL (normal range 0.068-0.115).
Based on the results of clinical investigations and according to the current consensus criteria for the clinical diagnosis of AD ( McKhann et al., 2011 ), the patient was diagnosed as multidomain-MCI due to AD.
The patient underwent a further formal cognitive assessment in her follow-up -1 year after the previous neuropsychological examination -that documented a rapid and significant worsening in almost all the cognitive domains, promoting the transition from amnesic multidomain MCI to dementia (Supplementary Table 1).
A pharmacologic treatment with Donepezil (10 mg/die) is ongoing.

Genetic findings and in silico predictions
Sanger sequencing of PSEN1 gene (NM_0 0 0 021.4) revealed the presence of a T to G substitution in exon 7 (c.638T > G) causing the replacement of the Isoleucine residue in position 213 with a Serine (p.I213S) ( Fig. 1 B). No other pathogenic variants were found in APP, PSEN1 and PSEN2 genes. I213S was not found in GnomADv2.1.1 database and Exome Variant Server. The ApoE genotype of the patient was ɛ 3/ ɛ 3 A more extensive genetic analysis was carried out on proband's genomic DNA to check the presence of additional genetic variants in the genes most frequently correlated with dementia. The study confirmed the presence of the PSEN1 I213S and excluded other variants of possible pathogenic significance.
The I213S substitution was predicted (1) to be deleterious by PROVEAN (score -5.669), (2) to affect the protein function by SIFT (score 0.0 0 0), (3) to be probably damaging by Polyphen-2 (score 1.0 0 0), and (4) to be disease causing by Mutation Taster (score 142) ( Table 1 ). Noteworthy, the I213S mutation showed the highest PROVEAN score among all the genetic variants at codon 213 of PSEN1 reported until now in association with AD ( Table 1 and 2 ). The residue 213 is highly conserved among different species ( Fig. 1 C), suggesting its relevant role for the function of presenilin 1 protein.

Discussion
We found a novel mutation (I213S) in PSEN1 gene in a young woman who developed an EOAD. Three other members in her family also had an early onset dementia, suggesting a genetically inherited form of AD with an autosomal dominant pattern, and a median age at onset of 35-40 years.
Several data strongly support the pathogenic nature of this novel mutation. The I-to-S substitution at codon 213 is a nonconservative replacement with potential impact on protein function ( Zhang, 20 0 0 ) taking into account that the native residue (i.e., Isoleucine) is a nonpolar aliphatic amino acid while the mutated residue (i.e., Serine) is a polar hydroxyl-containing amino acid, which is considered the third most disorder-promoting residue after Proline and Glutamine ( Vacic et al., 2007 ). Moreover, its short side chain, and the ability to form a hydrogen bond with the protein backbone confer to Serine the ability to mimic Proline ( Uversky, 2015 ).
Moreover, in silico predictions provided evidence in favor of the pathogenic role of the I213S variant, based on its localization in a dense hotspot region and its potential high impact on structure and function of PSEN1.
PSEN1 is composed of 9 transmembrane domains (TMDs) ( Li et al., 2014 ;Wolfe et al., 1999 ). During assembly of γ -secretase, PSEN1 undergoes an autocatalytic cleavage with the generation of an N-terminal fragment (comprising TMDs 1-6), and a Cterminal fragment (encompassing TMDs 7-9) ( Thinakaran et al., 1996 ;Wolfe, 2013 ). Interestingly, the PSEN1 mutations causing AD are mostly sited in 2 hotspots, each located at the inner core of a 4-TM bundle involved in the APP-binding and catalytic domain of γ -secretase. The first hotspot involves TMDs 2-5, the second one comprises TMDs 6-9 and affects residues in the immediate neighborhood of the catalytic residues Asp257 and Asp385 ( Bai et al., 2015 ). Mutations in these hotspots interfere with protease activity of γ -secretase increasing the production of A β42 or the A β42/A β40 ratio ( Bai et al., 2015 ;Bialopiotrowicz et al., 2012 ;Chavez-Gutierrez et al., 2012 ;Shimojo et al., 2008 ;Sun et al., 2017 ). Intriguingly, recent studies on the structural properties of PSEN1 and the γ -secretase complex by cryo-EM showed that the I213 residue in PSEN1 has its side chains pointing into the inner face of the APP-binding pore of γ -secretase, and that amino acid changes on this codon-as well as on other 18 residues located in this domain that are targeted for recurring mutations in AD patients -are likely to alter interactions with the substrate and/or hinder conformational changes induced by substrate binding ( Zhou et al., 2019 ), potentially leading to imbalance in the generation of A β peptides in favor of the longer species.
In this view, it's remarkable that 3 pathogenic mutations have already been described at the codon 213 of PSEN1 ( Tables 1 and 2 ), defining it as a true mutational hotspot.
In this group, the I213T was the best characterized genetic variant ( Table 2 ). It was described as a pathogenic genetic defect associated with EOAD in which typical signs and symptoms of AD were observed at the age of 45 ± 4.2 years ( Hardy, 1997 ;Kamino et al., 1996 ). The mutation was predicted to be deleterious for the structure and function of PSEN1 by in silico algorithms ( Han et al., 2020 ) ( Table 1 ). The pathogenic role of the I213T variant was supported by an enzymatic analysis of γ -secretase derived from I213T mutant PS1-expressing PSEN1/PSEN2-deficient (PS − / − ) cells and in cell-free assays demonstrating that the mutation specifically reduces A β40 levels, leading to an increased A β42/A β40 ratio, and at the same time increases the generation of longer A β species such as A β43, A β45, and A β46 ( Chavez-Gutierrez et al., 2012 ;Shimojo et al., 2008 ). In a 'knock-in' mouse model with the I213T mutation, elevated levels of the highly amyloidogenic A β42(43) peptide, but not A β40 peptide, were detected inside the brain by the age of 16-20 weeks ( Nakano et al., 1999 ). Moreover, the I213T knock-in mice exhibited tau pathology in the absence of A β deposition, indicating that the I213T change in the PSEN1 amino acid sequence causes AD not only by enhancing A β42 production but also by accelerating the formation and accumulation of neurofibrillary tangles in the brain ( Tanemura et al., 2006 ). Additional pathogenic effects involving inhibition of the neuroprotective functions of trophic factors eB1 and BDNF were more recently suggested for the I213T mutation ( Al Rahim et al., 2020 ).
Another genetic variant at the same codon of PSEN1 (I213L) was reported in several patients with EOAD in at least 2 unrelated families ( Rogaeva et al., 2001 ). Affected members in a Polish family developed the disease before their 50th and had progressive cognitive impairments characterized by memory loss at onset, followed by full dementia with aphasia, agnosia, apraxia, agraphia, acalculia, and extrapyramidal syndrome. Myoclonus was reported in 2 individuals ( Gołąb-Janowska M et al., 2010 ). In cell-free assays, the I213L mutation was found to radically increase A β42 production resulting in an elevated A β42/A β40 ratio ( Table 2 ) ( Bai et al., 2015 ;Sun et al., 2017 ).
A third genetic variant at codon 213 of PSEN1 (I213F) was identified in a Polish man with EOAD and a family history of probable AD. The proband's clinical picture was characterized by memory impairment as first symptom, at age 33, associated with extrapyramidal syndrome, and mood abnormalities. The onset of dis-ease in the other affected family members was also before 35 years ( Zekanowski et al., 2003 ). The I213F genetic variant was reported to increase A β42/A β40 ratio in a cell model of AD ( Table 2 ) ( Bialopiotrowicz et al., 2012 ).
Similar to the other genetic variants identified at codon 213 of PSEN1 gene, the clinical phenotype of the I213S carrier reported in our study substantially overlapped the classic phenotype of sporadic AD, characterized by a prevalent amnesic presentation of the disease followed by a progressive extension of the cognitive deterioration to other non-amnesic domains (Supplementary Table 1).
In line with all the functional studies performed on the other mutations at codon 213 of PSEN1 , the pathogenic value of the I213S mutation in AD needs to be confirmed by future in-depth mechanistic studies.
In a more general view, although PSEN1 mutations only explain a very low percentage of total AD cases, the understanding of the molecular mechanisms linking these genetic variations to the disease still deserves in-depth investigations because it may provide new insights to improve the design and development of therapeutic approaches for all the AD forms, including the most recurrent sporadic cases ( Van Cauwenberghe et al., 2016 ).

Disclosure statement
The authors have no actual or potential conflicts of interest.

Funding
This work was supported by Current Research Program from Italian Ministry of Health (RC 2018-21) to GDF, MC and GR, and Ministry of Health -5 × 10 0 0/2017 to GDF.
Authors declare that data contained in the manuscript being submitted have not been previously published, have not been submitted elsewhere, and will not be submitted elsewhere while under consideration at Neurobiology of Aging.
All authors have reviewed the contents of the manuscript being submitted, approved its contents and validated the accuracy of the data.

Declaration of Competing Interest
None