Causative Mutations and Genetic Risk Factors in Sporadic Early Onset Alzheimer’s Disease Before 51 Years

Abstract

Background:

Pathogenic variants in the autosomal dominant genes PSEN1, PSEN2, or APP, APOE4 alleles, and rare variants within TREM2, SORL1, and ABCA7 contribute to early-onset Alzheimer’s disease (EOAD). However, sporadic EOAD patients have been insufficiently studied to define the probability of being a carrier of one of these variants.

Objective:

To describe the proportion of each genetic variation among patients with very young-onset sporadic AD.

Methods:

We first screened PSEN1, PSEN2, and APP in 154 EOAD patients with an onset before 51 years and a negative family history. Among 99 patients with no mutation (NMC), whole exome sequencing (WES) was performed. We analyzed the APOE genotype and rare protein-truncating or missense predicted damaging variants of TREM2, SORL1, and ABCA7. Neurological examination and cerebrospinal fluid (CSF) biomarkers were systematically retrieved.

Results:

Nineteen (12.3%) mutation carriers (MC) harbored an APP or PSEN1 pathogenic or likely pathogenic variant. Among the NMC, 54/99 carried at least one genetic risk factor, including 9 APOE4/E4 homozygous, 37 APOE4 heterozygous, and 14 with a rare variant in another risk factor gene: 3 SORL1, 4 TREM2, and 9 ABCA7. MC presented an earlier disease onset (p < 0.0001) and associated neurologic symptoms more frequently (p < 0.002). All but one patient had at least 2 CSF biomarkers in abnormal ranges.

Conclusion:

The genetic component of very early sporadic EOAD gathers a substantial proportion of pathogenic variants in autosomal dominant genes and an even higher proportion of patients carrying genetic risk factors, suggesting an oligogenic determinism, even at this range of ages.

INTRODUCTION

Alzheimer’s disease (AD) is the leading cause of dementia. Although a genetic contribution to the etiology of early-onset AD (EOAD, onset before 66 years) is well documented, autosomal dominant inheritance due to pathogenic variants in the three genes APP, PSEN1, and PSEN2 [1 –3] is observed in a small minority of patients, more specifically in families with multiple generations affected by EOAD [4]. In other EOAD patients, the diagnostic yield has been reported to be very low, i.e., less than 3% overall [5]. In patients negatively screened for the autosomal dominant genes, moderate-to-high effect risk factors have been shown to contribute significantly to the genetic determinism of EOAD. Among them, the common APOE4/E4 genotype and the much more common APOE3/E4 genotype are associated with high odds ratios (ORs of 14.9 and 3.2, respectively, in Caucasian populations) and rare damaging variants in the TREM2, SORL1, and ABCA7 genes have been recently reported with respectively with ORs of 6.30 [2.86–13.86], 3.41 [2.09–5.55], and 2.54 [1.72–3.76] following gene-based analyses in the French population [6, 7].

In the context of the development of genomic medicine and facilitated access to genomic screens, patients and families are requesting information to their medical referents regarding genetic risks. Although multiple-generation EOAD families are highly suggestive of an autosomal dominant transmission, allowing clinicians to anticipate a putative identification of a pathogenic variant with the patients’ families, little is known about diagnostic yields in very young patients with a negative family history. We previously reported a 13% rate of pathogenic or likely pathogenic variants in the autosomal dominant genes in patients with sporadic EOAD and first symptoms before the age of 51, most of them could be documented as having occurred de novo in the patient [8]. However, such a young age of onset suggests a strong genetic contribution even in the negatively screened patients despite negative family history, not allowing to firmly reassure the offspring regarding their own risk. A small minority of these patients may harbor de novo mutations in novel genes such as VPS35 and MARK4 [9], the penetrance of which remains unknown. The role of genetic risk factors remains unclear in this specific population while the disease is considered to be highly related to genetic factors. The identification of one or multiple genetic factors may contribute to increase the likelihood that the genetic substratum of EOAD in a given patient is eventually oligogenic/multifactorial, hence providing arguments against pure autosomal dominant transmission. In addition, it might reinforce the certitude of the diagnosis, making unlikely a differential diagnosis causing early-onset dementia.

In previous studies, results on risk factors were generally presented as odds ratios among late-onset and early-onset AD patients. With the aim to help clinicians to inform patients and families prior to any genetic test request, we sought to determine the proportion of patients with an age of onset before 51, carrying pathogenic mutations or strong risk factors. We previously reported the detection rate of pathogenic variants in APP, PSEN1, and PSEN2 in 129 patients with sporadic AD and an age of onset before 51 [8]. We gathered these 129 patients with 25 novel patients and took advantage from WES data in patients negatively screened for the autosomal dominant genes, to assess the detection rate of rare damaging variants in the novel risk factor genes TREM2, SORL1, and ABCA7 in addition to the APOE genotype. In addition, as atypical symptoms have been found to be enriched in autosomal dominant EOAD genes mutations carriers, we report here the phenotype associated with EOAD in this specific population and regarding the genetic variation related to the disease.

METHODS

Patients included in this study were all cases with probable or certain AD, referred to the National Reference Center for Young Alzheimer Patients between May 1991 and May 2017 (RBM 02–59 & GMAJ Study, EudraCT 2009-010884-18, NCT01622894) from a nationwide memory clinics network, with an age of onset before 51, and no dementia family history after pedigree investigation performed by interview of at least one close relative. The AD diagnosis was performed according to the National Institute on Aging–Alzheimer’s Association [10]. All patients or legal representatives gave informed written consent in the context of a study approved by the CPP (Comité de Protection des Personnes) Ile de France II ethics committee. Part of this series was involved in another study from our group [8]. We report here additional patients, the results of risk factor genes and associated phenotypes gathering previously published and novel patients.

Before the diagnosis, patients underwent a comprehensive clinical examination including personal medical and family history, neurological examination, neuropsychological assessment and some underwent magnetic cerebral imaging and lumbar puncture with cerebrospinal fluid (CSF) biomarker analysis. Neuroimaging data were extracted from the medical files of each patient and reviewed by ML and DW. Neuropathological examination, using standard procedures, was performed when possible.

CSF biomarkers analysis was carried out using a standardized procedure already published elsewhere [8]. For each biomarker, the analysis was performed in duplicate and a coefficient of variation less than 15% was considered as acceptable. In this case, the mean of the two measured values was taken as final result. When the coefficient of variation was above 15%, samples were reanalyzed in duplicate. The quality of the results was ensured by the use of validated standard operating procedures and internal quality controls as all centers were already involved in nationwide studies [11 –13]. We verified that the mean value did not significantly differ between each laboratory (data not shown). CSF biomarker profiles were interpreted as supporting AD diagnosis if one of these two conditions was fulfilled: either 1) decreased Aβ₄₂ level (N > 550 pg/ml) or Aβ₄₂/Aβ₄₀ ratio (N > 0.045 following [14]), together with increased Tau (N < 350 pg/mL) or P-Tau levels (N < 60); or 2) Innogenetic Amyloid Tau Index (IATI) and [P-Tau]/[Aβ₄₂] ratios were both abnormal (respectively <0.8 and >0.211 as already published elsewhere [4]).

Genomic DNA was isolated using Flexigen DNA kit (Qiagen) from whole blood according to standard procedures. The entire coding regions of PSEN1 and PSEN2 and exons 16 and 17 of the APP gene were sequenced as previously described [4]. APP duplications were assessed by Quantitative Multiplex PCR of Short fluorescent Fragment (QMPSF) as previously described [15]. We classified APP, PSEN1, and PSEN2 rare variants following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) recommendations and we retained likely pathogenic (class 4) and pathogenic variants (class 5) [16]. Variants of unknown significance (class 3) were reported only when the prior classification following the Guerreiro et al. algorithm [17] was at least possibly pathogenic, although without enough formal evidence to be classified as likely pathogenic following the ACMG-AMP recommendations. For clarity, we will use the word “mutation” gathering all three above-mentioned categories of variants.

APOE genotype was determined for each patient by Sanger sequencing. For a subset of the patients negatively screened for the autosomal dominant AD genes, WES was performed as part of a previous case-control study [7, 18]. Briefly, the exomes were performed with a 142x average coverage using the Illumina technology after capture using an Agilent SureSelect human all exon kit. Raw sequenced were processed using BWA and GATK software suites, variants were annotated using SNPEff/SNPSift. Copy number variants were assessed using the CANOES software. All patients were from French ancestry and we checked their European ancestry using principal component analyses. Further quality checks included DNA contamination assessment, relatedness, depth of coverage, and the quality of the variants (for detailed information on exome sequencing, pipelines, and quality controls, see [7]). We extracted all TREM2, SORL1, and ABCA7 rare variants in all coding exons and flanking intronic sequences with a minor allele frequency (MAF)<1% in the non-Finish European population of ExAC [19] that fulfilled our criteria for variant deleteriousness with a previously demonstrated significant association with EOAD risk: protein truncating variants (PTV), i.e., nonsense, splice site, and frameshift variants, and missense strictly damaging (SD) variants (predicted damaging by 3/3 bioinformatics tools among Polyphen2, SIFT, and Mutation Taster). All patients with WES data available were negatively screened for other gene variants causing other types of genetic cognitive decline (targeted analysis of the WES data): MAPT, GRN, VCP, SQSTM1, FUS, TARDBP, CHMP2B, LRRK2, SNCA, PINK1, NOTCH3, HTRA1, COL4A1, PRNP, DNMT1, ITM2B, SERPINI1, CSF1R, TYROBP [20].

Based on their genetic results, two groups of patients were created: the mutation carrier (MC) group gathering all PSEN1, PSEN2, or APP mutation carriers and APP duplication carriers, and the non-mutation carrier group when no mutation was identified, with the exclusion of patients with no WES available (Fig. 1). We divided into five sub-groups: the APOE4/E4 carrier group, the APOE4/EX carrier group (E4/EX, gathering E4/E3 and E4/E2 patients), the Rare Risk Factor Carrier group (RRFC) including SORL1, TREM2, and ABCA7 PTV and SD rare variants carriers, and finally the No Risk Factor carriers (NRFC). In addition, for some specific relevant analysis, we gathered within the same group with all patients carrying at least one risk factor (among APOE4 allele and rare PTV and SD variants in the risk factor genes). Since patients could carry multiple risk factors, the groups could overlap.

Fig.1

Description of the genetic screening of our series of sporadic EOAD patients before the age of 51. EOAD, early onset Alzheimer’s disease; QMPSF, quantitative multiplex PCR of short fluorescent fragment; WES, whole exome sequencing.

Statistical analyses were performed using XLSTAT software. Since no genotype-phenotype study could be made for patients with no causal mutation and no WES data, clinical, biological, neuroimaging, and neuropathological data were only analyzed for the MC and NMC groups. The proportions of the clinical parameters for each group of patients (gathering neuropsychological assessment to assess typical or atypical presentations of AD, and any somatic sign at neurological examination including pyramidal or extrapyramidal syndrome, cerebellar ataxia, dystonia, and history of seizure or vascular event) were compared using a Fisher’s exact test due to small sample sizes by pairwise comparisons between the MC and NMC groups with multiple testing adjustments. Qualitative data analyzed were clinical presentation and type of associated neurological symptoms (including paraparesis, epilepsy, vascular event, cerebellar ataxia). All quantitative variables as age of onset, CSF Aβ₄₂, Tau, and P-Tau levels in each group were compared using a Mann-Whitney U Test as the size of the MC group was too small to estimate a gaussian distribution. We compared phenotypic data between MC and NMC groups or between MC (meaning a total of two independent tests for qualitative data and 4 independent tests for quantitative data). If a significant difference was found, a subsequent test between MC and each NMC subgroups was performed by pairwise comparison. The significance was set at p < 0.008 using a corrected threshold after Bonferroni correction for multiple testing.

RESULTS

Genetic landscape in 154 patients with sporadic EOAD before 51 years

Following our report on the genetic screen of autosomal dominant AD genes in 129 patients with sporadic EOAD starting before the age of 51 [8], we included 25 additional patients in this study adding up to a total of 154 patients included. The mean age of onset for the whole group of patients was 47.3 years [25–50] and the sex ratio was 0.734 for women. An APOE4 allele was carried by 67 (43.5%) of them, including 14 (9.1%) being APOE4 homozygous. A targeted genetic screen of autosomal dominant genes was first performed in the 25 additional patients. In addition to the previously reported 18 mutation carriers (17 PSEN1 variants and one APP duplication), we identified a novel PSEN1 pathogenic variant carrier (ALZ-652; Table 1). In total, 19 patients (12.3%) harbored a variant that could be classified as pathogenic or likely pathogenic (n = 18) and a variant of unknown significance that was previously reported as possibly pathogenic following Guerreiro et al. algorithm (n = 1) [17] (Fig. 1).

Table 1

Mutation carrier demographic and clinical data

Code	Gene	APOE	cDNA	p.	ACMG-AMP	De novo	AOO (year)	Disease duration (year)	Clinical presentation	MMSE	Aβ₄₂ (ng/L) N > 550	Tau (ng/L) N < 350	P-Tau N < 60	Symptom onset to MRI (year)	MRI	PET/SPECT Functional imaging
EXT -0502	PSEN1	3-3	c.331G>T	p.Gly111Trp	4	Y	47	4	typical	11	NA	NA	NA	3.0	diffuse cortical atrophy	fronto-parietal hypoperfusion with right predominance
EXT-851	PSEN1	2-3	c.350C>A	p.Pro117Gln	5	Y	37	2	typical	NA	587	>1200	173	2.0	diffuse cortical atrophy	NA
ALZ-0034	PSEN1	3-3	c.416T>A	p.Met139Lys	5	Y	37	10	typical	20	615	1204	129	5.3	diffuse cortical atrophy	temporo-occipito-parietal hypoperfusion
EXT-0670	PSEN1	2-4	c.428T>C	p.Ile143Thr	5	censoring, parental DNA NA	35	6	typical, paraparesis	15	393	1322	84	6.0	diffuse cortical atrophy	temporo-parietal bilateral hypometabolism
EXT-1242	PSEN1	3-3	c.488A>G	p.His163Arg	5	Y	34	4	frontal variant, myoclonic seizures	NA	615	1204	129	2.0	no cortical atrophy	frontal and temporo-parietal hypometabolism with right predominance
EXT-0149	PSEN1	3-3	c.518T>G	p.Leu173Trp	5	Y	34	4	frontal variant, paraparesis	NA	NA	NA	NA	2.9	bilateral moderate atrophy in the temporo-frontal region and the hippocampus	parieto-temporo-occipital hypoperfusion with left predominance
CAE-0007	PSEN1	4-4	c.539T>A	p.Ile180Asn	4	censoring, parental DNA NA	50	6	typical	14	NA	NA	NA	NA	NA	NA
EXT-0177	PSEN1	2-3	c.614_616del	p.Phe205_Gly206delinsCys	5	Y	42	7	typical	12	376	397	91	2.0	bilateral temporofrontal atrophy	bilateral occipito-temporal hypoperfusion
ROU-1551	PSEN1	3-4	c.650G>A	p.Gly217Asp	5	censoring, parental DNA NA	50	5	typical	21	NA	NA	NA	1.6	diffuse cortical atrophy with bilateral parietal predominance	parieto-temporo-occipital hypometabolism with left predominance
EXT -807	PSEN1	2-3	c.666G>C	p.Gln222His	4	censoring, parental DNA NA	46	4	typical, paraparesis	5	502	1000	132	4.0	diffuse cortical atrophy with moderate leukopathy	bilateral hypoperfusion predominant in the right frontal and temporal cortex
EXT-0680	PSEN1	2-3	c.691G>A	p.Ala231Thr	4	censoring, parental DNA NA	50	3	typical	NA	772	1028	113	NA	NA	NA
MON-0001	PSEN1	3-3	c.699G>C	p.Met233Ile	5	Y	28	8	typical, paraparesis, cerebellar ataxia	14	NA	NA	NA	2.7	hippocampal atrophy	NA
ROU-0128	PSEN1	3-3	c.710T>G	p.Phe237Cys	4	no parental DNA	25	15	typical	10	NA	NA	NA	NA	NA	parieto-temporo-frontal hypoperfusion
EXT-0504	PSEN1	3-3	c.722T>G	p.Leu241Arg	4	censoring, parental DNA NA	44	3	typical	11	464	595	94	2.7	bilateral temporoparietal atrophy	bilateral posterior associative area hypoperfusion with frontal and temporal extension
EXT-0235	PSEN1	2-3	c.869-2A>G	p.Ser290_Ser319delinsCys (Δ9)	5	Y	46	7	cortico-basal syndrome, myoclonic seizures	22	481	777	96	6.5	bilateral parietal atrophy	bilateral hypometabolism in the frontal, parietal and temporal associative cortex
SAL-0629	PSEN1	3-3	c.1078G>A	p.Ala360Thr	3	no parental DNA	45	7	posterior cortical atrophy	NA	487	217	35	6.7	hippocampal atrophy with mild non-specific leukopathy	NA
ALZ-652	PSEN1	3-3	c.1254G>T	p.Leu418Phe	5	censoring, parental DNA NA	35	8	frontal variant	7	475	735	115	0	moderate bilateral parietal atrophy	left parietal hypoperfusion
ROU-1306	PSEN1	3-3	c.1254G>T	p.Leu418Phe	5	Y	33	11	typical	19	248	1768	236	10.2	diffuse cortical atrophy with moderate leukopathy	NA
EXT-773	APP DUP	3-3	dup APP (7.6 Mb)	dup APP	5	Y	44	13	typical	NA	241	397	83	NA	intracerebral hemorrahge in the right temporal lobe	hypoperfusion in the posterior associative areas with right predominance

AOO, age of onset; MMSE, Mini-Mental State Examination; MRI, magnetic resonance imaging; NA, not available; Y, presence of a de novo mutation. Reference transcript used for PSEN1 variants nomenclature: NM_000021.3.

Among these 19 patients, pedigree assessment suggested a censoring effect in 7 (i.e., at least one unknown parent or early death from another cause than a neurological disorder). Among the 12 remaining patients, we previously showed that the mutation occurred de novo in all 10 patients for whom parental DNA was available [8, 9] (Fig. 1).

In the NMC group gathering the remaining 135 patients, WES data was available for 99 patients, allowing for the assessment of rare predicted damaging variants of TREM2, SORL1, and ABCA7 in addition to the APOE genotype. For 36 patients, DNA sample quality was insufficient to perform exome analysis. Among the 99 patients, 46 (46.9%) carried at least one APOE4 allele including 9 (9.1%) being homozygous. Four (4.0%) patients carried a TREM2, 3 (3.0%) a SORL1, and 9 (9.1%) an ABCA7 PTV or missense SD variant (Table 2). In total, 14 (14.1%) patients carried at least one of these variants, as two patients carried two rare variants, a SORL1 SD and a TREM2 SD variant in patient EXT-047 and a TREM2 SD and an ABCA7 PTV in patient EXT-1218 (Table 2 and Fig. 2). In addition, the latter patient carried an APOE4/E4 genotype. In total, among the 99 patients from the NMC group, 54 (54.5%) carried at least one risk allele including 8 (8.1%) carrying multiple risk alleles (Table 2). This number reached 22 (22.2%) if APOE4/E4 genotypes were considered as two distinct risk alleles. Among the rare variant carriers, DNA of unaffected elderly parents was available for three and all three variants were within ABCA7 gene and were inherited from an asymptomatic parent.

Fig.2

Clinical phenotype distribution of the 99 patients negative for causative mutation and WES analyzed. EAOD, early onset Alzheimer’s disease; WES, whole exome sequencing; APOE4/EX, heterozygous APOE4 carrier; incl., including.

Table 2

Rare risk factor carrier demographic and clinical data

Code	APOE	Rare Risk Factors Gene(s)	cDNA	p.	SD/PTV	gnomAD NFE non Neuro v2.1 Frequency	Pedigree information	AOO (year)	Disease duration (year)	Clinical presentation	MMSE	Aβ₄₂ (ng/L) N > 550	Tau (ng/L) N < 350	P-Tau (ng/L) N < 60	Symptom MRI onset to MRI (year)	MRI	PET/SPECT Functional imaging	Multiple risk factors
EXT-1185	3-4	TREM2	c.92C>T	p.Ser31Phe	SD	0.00002975	parental DNA NA	48	11	typical	NA	224	454	NA	10.4	bilateral hippocampal atrophy	NA	x
EXT-1218	4-4	TREM2	c.140G>A	p.Arg47His	SD	0.002504	parental DNA NA	50	4	typical	20	760	1078	137	3.2	occipitoparietal atrophy	inferior temporo-parietal cortex, precuneus and posterior cingulum hypometabolism	x
ABCA7	c.67-1G>A	p.?	PTV	0.0002817
ROU-168	3-3	TREM2	c.140G>A	p.Arg47His	SD	0.002504	parental DNA NA, censoring	48	4	typical, extrapyramidal syndrome	9	NA	NA	NA	NA	NA	NA
EXT-047	3-3	TREM2	c.140G>A	p.Arg47His	SD	0.002504	parental DNA NA	47	2	frontal variant	NA	241	425	57	2.0	left temporal and right parietal atrophy	bilateral temporo-occipital hypoperfusion	x
SORL1	c.4166A>T	p.Asp1389Val	SD	0
ALZ-532	3-3	SORL1	c.372C>A	p.Ser124Arg	SD	0	parental DNA NA	48	2	typical	17	242	546	110	NA	NA	NA
EXT-987	3-3	SORL1	c.1805C>T	p.Ser602Leu	SD	0	parental DNA NA, censoring	46	3	typical	20	546	354	71	NA	NA	NA
ALZ-432	3-3	ABCA7	c.2026G>C	p.Ala676Pro	SD	0	parental DNA NA, censoring	48	4	typical	19	NA	NA	NA	NA	NA	left parietotemporal hypoperfusion
EXT-1120	3-4	ABCA7	c.3577+1G>C	p.?	PTV	0.0000337	Inherited from asymptomatic parent	47	7	primary progressive aphasia	4	624	1106	122	6.8	diffuse cortical atrophy with bilateral frontal and hippocampal predominance	bilateral fronto-parietal hypometabolism	x
EXT-1135	4-4	ABCA7	c.302T>G	p.Leu101Arg	SD	0.001607	parental DNA NA	48	3	typical	6	720	1230	129	2.5	fronto-temporal atrophy	NA	x
ROU-1322	3-3	ABCA7	c.5794C>T	p.Arg1932Cys	SD	0.00005945	inherited from asymptomatic parent	50	7	typical	22	573	624	100	NA	NA	NA
ALZ-475	2-4	ABCA7	c.991dupC	p.Leu331fs	PTV	0	parental DNA NA, censoring	46	5	typical	6	373	369	64	NA	NA	bilateral posterior associative areas hypoperfusion	x
EXT-1102	3-4	ABCA7	c.4878T>A	p.Tyr1626*	PTV	0	parental DNA NA, censoring	48	11	typical	NA	NA	NA	NA	0.7	normal	temporoparietal hyopoperfusion with left predominance	x
EXT-573	3-3	ABCA7	c.4208delT	p.Leu1403fs	PTV	0.001328	inherited from asymptomatic parent	45	1	typical	26	390	659	27	NA	NA	NA
EXT-697	4-4	ABCA7	c.4208delT	p.Leu1403fs	PTV	0.001328	parental DNA NA, censoring	49	13	typical	26	685	627	80	12.3	diffuse cortical atrophy	posterior associative regions hypometabolism with right predominance	x

SD, missense strictly damaging variant (i.e., predicted damaging by all three bioinformatics tools: SIFT, Mutation Taster, and PolyPhen2); PTV, protein truncating variant (i.e., introducing a premature stop codon); AOO, age of onset; MMSE, Mini-Mental State Examination; MRI, magnetic resonance imaging. Reference transcripts used for variant nomenclature: NM_003105.5 (SORL1), NM_018965.2 (TREM2) and NM_019112.3 (ABCA7).

Phenotypic spectrum associated with autosomal dominant and risk factor genes

The average age of onset (AOO) was significantly earlier in the MC (40.1 years±7.6) compared to the NMC group (48.2±1.6) (p < 0.0001). When comparing MC with each of the NMC subgroups, AOO was still significantly lower in the MC group (p < 0.0001). There was no significant difference of AOO among NMC subgroups (Supplementary Table 1).

Considering the MC group and the 99 NMC patients, a typical AD phenotype (memory disturbances, apraxia, and impairment of spatial skills) was the most frequent clinical presentation, observed in 14 (77.8%) PSEN1, the APP duplication, 7 (77.8%) APOE4/E4 and 30 (81.8%) APOE4/EX carriers (Fig. 2 and Supplementary Table 1). Among MC and NMC groups, 23 (23.2%) patients presented atypical signs: 2 PSEN1 MC (p.His163Arg and p.Leu418Phe) suffered from behavioral disorders compatible with a frontal presentation at 34 and 35 years of age, respectively. The patient with the PSEN1 p.Ala360Thr variant had a cortical posterior atrophy and the patient harboring the p.Ser290_Ser319delinsCys (Δ9) variant had symptoms suggestive of cortico-basal syndrome starting at the age of 46. There was no logopenic aphasia among mutation carriers. One APOE4/E3 patient presented a parietal syndrome and another one had a cortico-basal syndrome. The patient carrying a SORL1 and a TREM2 SD variant exhibited a frontal presentation. Three patients carrying an APOE4/E3 genotype had predominant language impairment suggestive of a logopenic aphasia and one of them was also carrying an ABCA7 variant. Among the NRFC group, 13 (28.9%) had atypical symptoms: 2 with a frontal presentation, 3 with cortical posterior atrophy, 4 with logopenic aphasia, 3 a parietal syndrome, and 1 with a cortico-basal syndrome (Fig. 2).

Associated neurological symptoms were more frequent in the MC group compared to NMC (p = 0.002) (Table 3 and Supplementary Table 1). Considering all MC and NMC, 27 patients (19.4%) had associated neurological symptoms occurred early in the disease. Eighteen patients (15.3%) experienced seizures (including absence seizures, myoclonic seizures, focal and generalized seizures) during disease course but only one PSEN1 patient had generalized seizures 9 years before onset. Six patients presented spastic paraparesis, including 4 carrying a PSEN1 MC (p.Ile143Thr, p.Leu173Trp, p.Gln222His, p.Ser290_Ser319delinsCys (Δ9)), one APOE4/E3 patient, and one patient with no risk factor. Cerebellar ataxia was observed in only two PSEN1 variant carriers (p.Met233Ile and p.Ser290_Ser319delinsCys (Δ9)). There was no significant difference between subgroups regarding neurological associated symptoms.

Table 3

Clinical and biological phenotypic analysis

	MC (n = 19)	NMC (n = 99)	p
AOO (years)	40.1±7.6	48.2±1.6	p < 0.0001 (1)
Typical presentation (n = (%))	15 (78.9%)	72 (72.7%)	p = 0.777 (2)
Atypical presentation including:	4 (21.1%)	18 (18.3%)
- frontal variant	2 (10.5%)	3 (3.0%)
- posterior variant	1 (5.3%)	3 (3.0%)
- logopenic variant	0	6 (6.1%)
- parietal variant	0	4 (4.1%)
- cortico-basal syndrome	1 (5.3%)	2 (2.0%)
NA	0	9 (9.1%)
Associated neurological symptoms including:	10 (52.6%)	17 (17.2%)	p < 0.002 (2)
- paraparesis	4	2
- epilepsy	4	14
- vascular event	1	1
- cerebellar ataxia	2	0
AD Biomarkers	13 (68.4%)	77 (77.8%)
- Aβ₄₂ (ng/L) [min-max]	491.0 [241–772]	461.3 [179–902]	p = 0.279 (1)
- Tau (ng/L) [min-max]	901.2 [217–1768]	713.6 [227–1302]	p = 0.132 (1)
- P Tau (ng/L) [min-max]	118.0 [35–238]	99.3 [27–397]	p = 0.086 (1)
All CSF biomarkers within pathologic ranges	9 (47.4%)	57 (57.6%)
CSF profile supporting AD diagnosis^*	12 (63.1%)	77 (77.8%)

MC, mutation carriers; NMC, non-mutation carriers; AOO, age of onset; NA, not available. 1) Mann-Whitney test; 2) Fisher’s exact test; all statistical tests were performed considering a significant p-value if less than a corrected threshold of 0.008 after Bonferroni correction for multiple testing. “Typical presentation” refers to amnestic symptoms (memory disturbances, apraxia, and impairment of spatial skills). “Atypical presentation” refers to patients with all possible other presentations not compatible with the typical presentation. “Associated neurological symptoms” refers to symptoms that could be associated either with typical or atypical presentations. ^*either (i) decreased Aβ₄₂ level (N > 550 pg/ml) or Aβ₄₂/Aβ₄₀ ratio (N > 0.045), together with increased Tau (N < 350 pg/mL) or P-Tau levels (N < 60); or (ii) IATI and [P-Tau]/[A β₄₂] ratios were both abnormal (respectively <0.8 and >0.211).

Anatomical brain imaging was available for 86 patients including 15 PSEN1 MC and the APP duplication carrier. Atrophy was reported for 64 (74.4%) of them without any significant difference between MC and NMC groups. Three patients exhibited microbleeds, including two patients with diffuse microbleeds (one PSEN1 (p.Gln222His) MC and the APP duplication carrier). Three patients suffered from cerebrovascular events: one SORL1 variant carrier presented an ischemic stroke on MRI, the APP duplication carrier had an intracerebral haemorrhage in the right temporal lobe 4 years after onset while taking anticoagulant therapy, and one from the NRFC group presented a cerebral thrombophlebitis of the venous longitudinal upper sinus 2 years after onset.

CSF biomarker analyses were performed in 90 (58.4%) patients. Among them, 69 patients (76.7%) presented the association of amyloid and Tau or P-Tau biomarkers within pathological ranges. For the 21 remaining samples, using IATI, P-Tau/Aβ₄₂ ratio and the Aβ₄₂/Aβ₄₀ ratio, 20 patient CSF were considered as compatible with AD [4]. Interestingly, the PSEN1 p.Ala360Thr variant carrier with cortical posterior atrophy had only one abnormal value (decreased Aβ₄₂ level) and did not meet our biological criteria. Of note, this is the only PSEN1 variant in the current series requiring more genetic or functional evidence for pathogenicity to definitely conclude.

Neuropathological examinations were available for 7 patients (2 biopsies, 5 autopsies). Aβ extracellular deposits and neurofibrillary tangles were moderate to severe in large cerebral areas for all samples. Aβ deposits were less common in the cerebellum and the brainstem. Large cotton-wool plaques were found in the brain biopsy of the p.Leu173Trp PSEN1 variant carrier who presented a frontal variant and spastic paraparesis. Lewy body deposits were frequent and diffuse for 2 PSEN1 MC (p.Leu173Trp and p.Ser290_Ser319delinsCys (Δ9)) and the APP duplication carrier. Diffuse cerebral amyloid angiopathy was noticed in 5 patients including 3 PSEN1 variant carriers (p.Ser290_Ser319delinsCys (Δ9), p.Leu173Trp, p.Met233Ile) and the APP duplication carrier.

DISCUSSION

In addition to the contribution of (likely) pathogenic variants in autosomal dominant genes which were present in 12.3% of our series of 154 patients with sporadic EOAD starting before 51, this study showed that 54.5% of the 99 remaining patients assessed by WES exhibited at least one genetic risk factor considered as increasing moderately to highly the risk of EOAD. Our study brings arguments supporting a larger genetic screening in these patients, instead of a narrowed screen focused on the 3 autosomal dominant genes. However, no genetic counselling can be performed based on risk factor results. Gathering all genetic variants carried by sporadic EOAD patients before 51 years in this study, our data suggest that a genetic screen may identify 1) highly penetrant mutations in a substantial proportion of patients with critical consequences for genetic counselling— especially for the offspring given the de novo occurrence of most of these mutations (if not all), and 2) one or even multiple genetic risk factors in an even larger proportion of patients, supporting the hypothesis of a multifactorial determinism of the disease and providing positive arguments against the presence of a putatively highly penetrant single genetic variant.

A limited number of studies have described the implication of the three autosomal dominant genes in sporadic EOAD. They reported a lower rate of pathogenic variants (6.5% on average) but the cohorts included patients with an AOO up to 65 [21 –24]. Therefore, the very low rate of pathogenic variants as compared to sporadic AD patients with onset after 51 probably explains this discrepancy.

Interestingly, among the NMC patient data, 8.1% had multiple risk alleles, providing formal evidence of a multifactorial determinism in these patients. Up to now, rare damaging variants of TREM2, SORL1, and ABCA7 are considered as genetic risk factors [7, 18] and should not be used in genetic counselling in asymptomatic relatives. Among three cases with a rare variant and parental DNA available, 3 carried an ABCA7 variant that was inherited from one of their asymptomatic parents (aged between 68 and 93 years), including 2 PTV.

Of note, most of the association analyses obtained on TREM2, SORL1, and ABCA7 were performed at the gene level, providing exome-wide significance when collapsing PTV with rare missense, predicted damaging variants [7]. From this evidence, it is likely that any novel or previously reported PTV can be considered as a risk variant at the individual level with a high confidence. However, this does not hold true for all missense variants. Indeed, only a few recurrent variants (e.g., TREM2 p.Arg47His [25]) were associated with AD at the single variant level. We cannot exclude that some of the other missense variants, even if predicted damaging by bioinformatics tools, may not impair the protein function and be actual risk factors. We expect that a small minority of them may be neutral. For these variants, a formal evidence may be assessed by in vitro tests, as it is unlikely to have enough power for variant-based association analyses due to the extreme rarity of most of them.

In sporadic forms of neurodegenerative diseases, i.e., in absence of a positive family history, a few hypotheses may be assessed to explore a putative genetic contribution: 1) the de novo occurrence of highly penetrant variants with autosomal dominant transmission, 2) somatic variants affecting part of the body and more specifically the brain, 3) autosomal recessive inheritance, and 4) a complex determinism, in which oligogenic forms may be encountered when a few genes have a high impact (for review see [26]). Herein and in a previous report, we assessed the role of de novo mutations in autosomal dominant genes [8]. We also previously assessed the somatic mutation hypothesis and identified only a few cases with putatively damaging variants in SORL1 but not in the known autosomal dominant genes [27]. The case of autosomal recessive inheritance has been suggested to explain part of the genetic component in EOAD [28]. However, up to now, only very few families were reported with probable recessive inheritance of variants in APP (p.Glu693Δ and p.Arg673Val) [29]. Here, we show that oligogenic inheritance may be encountered in a significant proportion of sporadic cases.

Given the putatively atypical presentation of EOAD among some families with autosomal dominant inheritance, we evaluated the clinical presentations of EOAD in our series with sporadic patients with a very early onset and with no pathogenic mutation. Interestingly, 18.3% of them exhibited an atypical presentation, including frontal presentation, logopenic aphasia, cortical posterior atrophy, parietal presentation, and cortico-basal syndrome (3.0%, 6.1%, 3.0%, 4.1%, and 2.0%, respectively). This is consistent with the frequency of atypical manifestations in EOAD (22% to 64%) overall [30 –33]. As expected, MC exhibited a significantly younger AOO compared to NMC even in a very young onset AD series (before 51). They also had more frequent associated neurologic symptoms with almost a third of them having spastic paraparesis. This phenotype is now reported with more than 20 PSEN1 mutations [34 –38]. We found profuse cotton wool plaques in the biopsy from one patient with spastic paraparesis but complete neuropathological examination was not available to describe the distribution of amyloid and tau pathology.

One patient carrying both p.Arg47His TREM2 and p.Asp1389Val SORL1 variant had an initial frontal presentation but CSF biomarker profile supporting an AD diagnosis. TREM2 variants have been associated with frontotemporal dementia cases of recessive inheritance [39, 40]. We report in this study, the first ABCA7 variant (p.Tyr1626*) associated with logopenic aphasia. Interestingly, he also presented early seizures in accordance with the 10% of AD patients with seizures harboring an ABCA7 variant in a Belgian cohort [41]. Seizures are the most frequent associated symptom and sometimes occur early in the disease course of a mutation carrier [42]. However, only one PSEN1 patient presented seizures before cognitive symptoms in our series.

Fig.3

Decision algorithm for genetic assessment regarding sporadic EOAD patient before the age of 51. AD, Alzheimer disease; AOO, age of onset; WES, whole exome sequencing; WGS, whole genome sequencing.

Ninety-nine percent of the CSF biomarkers were compatible with AD with no specific profiles associated with MC consistent with a recent review that did not report any particular CSF profile associated with autosomal dominant EOAD [43]. Only one PSEN1 CSF sample remained atypical with a decreased Aβ₄₂ level but normal Tau and Phospho-Tau levels. This point raises the question of restraining the genetic analysis only to patients with all CSF biomarkers typically compatible with AD.

One strength of our study was the high number of the very early onset AD cases, recruited from a nationwide network using same standardized diagnostic criteria (National Reference Centre for young Onset AD guidelines). Following our results, a molecular diagnosis algorithm could be proposed to help clinicians decide when a patient with negative family history should be proposed to a genetic assessment (Fig. 3). Therefore, if all CSF AD biomarkers and ratios are within the normal ranges, there is no argument to firstly consider AD. Conversely, only one biomarker or ratio compatible with AD seem sufficient to propose genetic analysis in sporadic AD under the age of 51 at onset, not to miss a potential carrier.

Our study supports the recommendation of a genetic screening for every patient with sporadic EOAD with an AOO before 51, especially when AD diagnosis is made likely not only by clinical and imaging arguments, but also by at least one supportive CSF biomarker. We could identify up to 12% patients with a genetic cause among the autosomal dominant genes. Accurate identification of such variant carriers give the opportunity to propose an adequate genetic counselling in the family and inclusion in prevention trials such as DIAN-TU (Dominantly Inherited Alzheimer Network Trial Unit) [44]. In addition, a high proportion of the NMC exhibited at least one genetic risk factor among the moderate-to-high risk genetic factors identified to date. Taken together, this suggests that, even at that age, the etiology of AD is heterogeneous, with a monogenic inheritance in some patients and a complex etiology supported by several genetic factors in others. These critical pieces of information are required before any genetic test in patients as they can help families to get prepared to one or another outcome. Our results might also be of interest for the future set up of preventive trials regarding presence of single or multiple genetic risk factors.

Footnotes

ACKNOWLEDGMENTS

This study was funded by grants from the Clinical Research Hospital Program from the French Ministry of Health (GMAJ, PHRC, 2008/067), the CNR-MAJ, the JPND PERADES program.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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