Gael Nicolas
email: gaelnicolas@hotmail.com

Aline Zarea

Morgane Lacour

Olivier Quenez

Stephane Rousseau

Anne-Claire Richard

Antoine Bonnevalle

Catherine Schramm

Robert Olaso

Florian Sandron

Gaël Nicolas1

Aline Zaréa2

Morgane Lacour2

Olivier Quenez1

Stéphane Rousseau1

Anne-Claire Richard1

Antoine Bonnevalle1

Catherine Schramm1

Robert Olaso4

Florian Sandron4

Anne Boland4

Jean-François Deleuze4

Daniela Andriuta5

Pierre Anthony6

Sophie Auriacombe7

Anna- Chloé Balageas8

Guillaume Ballan9

Mélanie Barbay5

Yannick Bejot10

Serge Belliard11

Marie Benaiteau13

Karim Bennys14

Stéphanie Bombois15

Claire Boutoleau-Bretonniere17

Pierre Branger18

Jasmine Carlier13

Leslie Cartz-Piver19

Pascaline Cassagnaud20

Mathieu-Pierre Ceccaldi21

Valérie Chauvire22

Yaohua Chen23

Julien Cogez18

Emmanuel Cognat24

Fabienne Contegal-Callier26

Lea Corneille21

Philippe Couratier27

Benjamin Cretin28

Charlotte Crinquette20

Benjamin Dauriat29

Sophie Dautricourt30

Vincent De La Sayette18

Astrid De Liège32

Didier Deffond33

Florence Demurger34

Vincent Deramecourt23

Céline Derollez20

Elsa Dionet33

Martine Doco

Julien Dumurgier24

Anais Dutray38

Frédérique Etcharry-Bouyx22

Maïté Formaglio30

Audrey Gabelle14

Anne Gainche-Salmon12

Olivier Godefroy5

Mathilde Graber39

Chloé Gregoire40

Stephan Grimaldi21

Julien Gueniat39

Claude Gueriot21

Virginie Guillet- Pichon22

Sophie Haffen41

Cezara-Roxana Hanta12

Clemence Hardy2

Geoffroy Hautecloque6

Camille Heitz42

Claire Hourregue24

Thérèse Jonveaux44

Snejana Jurici46

Lejla Koric21

Pierre Krolak-Salmon30

Julien Lagarde48

Hélène-Marie Lanoiselée51

Brice Laurens40

Isabelle Le Ber15

Gwenaël Le Guyader52

Amélie Leblanc53

Thibaud Lebouvier23

Richard Levy15

Anaïs Lippi55

Marie-Anne Mackowiak20

Eloi Magnin56

Cecilia Marelli55

Olivier Martinaud18

Aurelien Maureille20

Raffaella Migliaccio15

Emilie Milongo-Rigal13

Sophie Mohr39

Helene Mollion30

Alexandre Morin1

Julia Nivelle58

Camille Noiray48

Pauline Olivieri48

Claire Paquet24

Jérémie Pariente13

Florence Pasquier23

Alexandre Perron5

Nathalie Philippi28

Vincent Planche7

Hélène Pouclet-Courtemanche60

Marie Rafiq13

Adeline Rollin-Sillaire20

Carole Roué-Jagot48

Dario Saracino15

Marie Sarazin48

Mathilde Sauvée61

François Sellal6

Marc Teichmann16

Christel Thauvin64

Quentin Thomas10

Camille Tisserand13

Cédric Turpinat55

Laurène Van Damme38

Olivier Vercruysse65

Nicolas Villain15

Nathalie Wagemann17

Camille Charbonnier66

David Wallon2

Gaël Nicolas

Assessment of Mendelian and risk factor genes in Alzheimer disease: a prospective nationwide clinical utility study

Keywords: Alzheimer disease, risk variant, pathogenic variant, exome, clinical utility

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Several countries are implementing genomic medicine plans in public healthcare, mostly for patients with rare diseases. Early-onset dementia is a clinical indication for exome/genome sequencing [START_REF] Martin | PanelApp crowdsources expert knowledge to establish consensus diagnostic gene panels[END_REF][START_REF] Levy | Genomic medicine 2025: France in the race for precision medicine[END_REF] . Early-onset Alzheimer Disease (EOAD, age of onset (AOO) <66 years) is the first cause of early-onset dementia [START_REF] Hendriks | Global Prevalence of Young-Onset Dementia: A Systematic Review and Metaanalysis[END_REF] and accounts for 5-10% of all Alzheimer Disease (AD) patients [START_REF] Sirkis | Dissecting the clinical heterogeneity of early-onset Alzheimer's disease[END_REF] . It thus represents a potentially large source of genetic screening requests and can be considered somewhere between rare and common diseases. Unlike many other rare early-onset neurodegenerative disorders, the cause of EOAD is not monogenic in the majority of cases [START_REF] Nicolas | From Common to Rare Variants: The Genetic Component of Alzheimer Disease[END_REF] . In addition, the clinical utility of exome/genome sequencing or even extended gene panels has not been assessed in large prospective AD series and it remains unclear which patients should be prioritized, if some should be.

Overall, pathogenic variants in APP, PSEN1 or PSEN2 causing monogenic forms of AD are encountered in less than 0.1% AD patients [START_REF] Nicolas | From Common to Rare Variants: The Genetic Component of Alzheimer Disease[END_REF] . They generally affect young patients with average ages of onset of 50.9, 44.4, and 53.9 years, respectively [START_REF] Lanoiselee | PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases[END_REF] . Among EOAD patients, a large diversity of pathogenic variant detection rates has been reported, depending on inclusion criteria 6-9 10,11 . Monogenic causes probably represent less than 20% of all EOAD cases. In non-monogenic AD, genetics factors are thought to play a significant role, as suggested by twin studies [START_REF] Gatz | Role of genes and environments for explaining Alzheimer disease[END_REF] . Most risk variants are shared between EOAD and late-onset AD (LOAD) cases, with diverse effects and frequencies. The most important AD genetic risk factor is the ε4 allele of the APOE gene, with a frequency of ~14% in individuals from European ancestry and AD lifetime risks of ~20% and ~50% at the age of 85, respectively for heterozygotes and homozygotes [START_REF] Van Der Lee | The effect of APOE and other common genetic variants on the onset of Alzheimer's disease and dementia: a community-based cohort study[END_REF] . In addition, significant progress has been made in the understanding of the genetics of complex forms of AD during the last decade. Large-scale case-control studies using either DNA chips [START_REF] Bellenguez | New insights into the genetic etiology of Alzheimer's disease and related dementias[END_REF] or exome/genome sequencing [START_REF] Holstege | Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease[END_REF] identified common and rare variants influencing the risk of AD with a broad spectrum of effects. Common variants are associated with low (<1.3) odds ratios (OR), precluding a clinical use, while polygenic risk scores (PRS), gathering the effect of multiple common variants seem to be primarily driven by the APOE genotype [START_REF] Van Der Lee | The effect of APOE and other common genetic variants on the onset of Alzheimer's disease and dementia: a community-based cohort study[END_REF] and miss rare variants. Rare coding variants with a larger effect (OR ranging from 1.5 to more than 27) have recently been reported, with a stronger effect among EOAD patients. Some of these rare risk variants, in the SORL1, TREM2, ABCA7, ABCA1 or ATP8B4 genes, may be worth using in a clinical setting [START_REF] Holstege | Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease[END_REF][START_REF] Nicolas | SORL1 rare variants: a major risk factor for familial early-onset Alzheimer's disease[END_REF][START_REF] Jonsson | Variant of TREM2 associated with the risk of Alzheimer's disease[END_REF][START_REF] Steinberg | Loss-of-function variants in ABCA7 confer risk of Alzheimer's disease[END_REF] . Indeed, clinical genetics interpretation is generally restricted to Mendelian genes. Yet, it may be seen as questioning to report a "negative" result to a patient who could elsewhere be heterozygote for a strong risk factor. Although risk variants cannot be used for genetic counseling, they still contribute to the disease etiology in patients with positive carrier status and thus represent a valuable source of information for patients and families seeking for responses to questions regarding disease etiology. The strongest EOAD rare risk factors appears to be loss-of-function (LOF) SORL1 variants, with an average OR of 27.5 [START_REF] Nicolas | SORL1 rare variants: a major risk factor for familial early-onset Alzheimer's disease[END_REF][START_REF] Raghavan | Whole-exome sequencing in 20,197 persons for rare variants in Alzheimer's disease[END_REF]20 . This even questioned whether SORL1 should be considered as an autosomal dominant AD gene [START_REF] Pottier | High frequency of potentially pathogenic SORL1 mutations in autosomal dominant early-onset Alzheimer disease[END_REF] , but we J o u r n a l P r e -p r o o f recently showed that penetrance is not complete by age 70, except in the context of APOE-ε4 homozygosity [START_REF] Schramm | Penetrance estimation of Alzheimer disease in SORL1 loss-of-function variant carriers using a family-based strategy and stratification by APOE genotypes[END_REF] . The case of other genes with rare risk variants is clearer from that perspective, given their lower OR compared to SORL1 LOF variants.

In a prospective study, called ECASCAD (Exome -Clinical Application of SequenCing in Alzheimer Disease), we included 700 AD patients from France and proposed exome sequencing as a first-tier genetic test. We interpreted rare variants in Mendelian dementia genes and built a clinical interpretation framework for AD-associated risk variants. Results were explained to patients and/or caregivers by referring clinicians following a personalized recommendation based on the genetic report. In absence of a monogenic cause, results of APOE genotype and rare risk factors were reported to the patients. We report the detection rate of autosomal dominant AD (likely) pathogenic variants, those in AD differential diagnoses, and risk factors, based on pedigree structure and AOO in probands.

Based on this prospective series as well as data from the literature and our own retrospective series, we propose recommendations on clinical indications for genetic screening.

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Methods

Patient enrollment and study procedures

Patients were included from 40 French centers (see supplementary information for detailed procedures). Diagnoses were based on IWG-2 criteria [START_REF] Dubois | Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria[END_REF] and an AD pathophysiological process was assessed by CSF AD biomarkers or amyloid PET if CSF was not available. Inclusion criteria were as follows: (i) a diagnosis of typical or atypical AD with AD pathophysiological process, (ii) first symptoms noticed at the age of 65 or before, whatever the family history, (iii) absence of an already known monogenic cause. Patients were not included in case of: (i) AD CSF biomarkers not consistent with an AD pathophysiological process, (ii) a differential diagnosis better explained the symptoms, (iii) previous genetic assessment using sequencing techniques, except for APOE genotyping (in case of known APOE genotype upon inclusion, patients were included regardless of the result), (iv) a likely pathogenic / pathogenic (LP/P) variant was already known in the family, and (v) if one of the parents was born in a non-European country or self-reported as being of non-European descent, as current knowledge on rare risk factors relies mainly on patients of European ancestry. In addition, five centers were allowed to include patients with AOOs until 75 years so that we could extend the clinical utility study to patients with LOAD (66-75 years). While in these age categories, we do not expect pathogenic variants in monogenic dementia genes, except a few variants with reduced penetrance, some of these patients may still have questions on disease etiology and/or family history, although genetic tests are usually not requested by the patients or not accepted by physicians, stating that there is no clinical indication.

Although this part of the study was not powerful enough to drive conclusions, we opened the inclusions with the aim to measure how many patients would request such an analysis, among LOAD patients, as an exploratory add-on to the study.

All patients or their legal representatives provided informed written consent for genetic analyses. This study was approved by the CPP-Ouest III ethics committee (notification 2018-A02359-46). Patients were included from December 2018, until n=700 inclusions were reached (June 2021).

Study procedures are presented in Figure 1 and in supplementary information. Briefly, patients underwent at least 2 visits, one upon inclusion, and one for the results delivery, by the same physician, who was also generally in charge of clinical follow-up. Specific information was provided before inclusion. To help the clinicians, a standardized and personalized information accompanied the genetic report. Templates of letters to referring physicians, key points summaries, and templates of letters to patients are available upon request (in French).

Clinical interpretation of variants

We chose to perform exome sequencing to access the coding sequence of a panel of genes with good sensitivity and specificity including single nucleotide variants (SNV), short insertions and J o u r n a l P r e -p r o o f deletions (indels) and Copy Number Variants (CNVs) and to allow the reinterpretation of existing data in the future in light of the discovery of novel genes without the need to resequence. Exome sequencing and bioinformatics pipelines are displayed in supplementary information and Figure 1.

Rare (allele frequency <1%) SNV, indels and CNVs in a list of 28 Mendelian dementia genes were interpreted following the American College of Medical Genetics and Genomics -Association for Molecular pathology (ACMG-AMP) recommendations [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF] by an expert geneticist. Variants classified as class 4 (likely pathogenic, LP) or class 5 (pathogenic, P) were returned to patients. Some of the class-3 variants (uncertain significance) were also returned, when additional investigations might help reclassifying them in the future.

Regarding risk factors, in the absence of existing consensus or interpretation guidelines, we built a framework for variant interpretation and classification (Figure 2 and supplementary information). After literature review, we first restricted the analysis to a list of validated AD-risk genes, which we defined as genes with exome-wide significance of a burden of rare, selected coding variants associated with AD or single rare variants with genome-wide significance and with at least one replication and OR>1.5. Then, we defined stringent criteria for classification as a definite AD risk factor and finally classified those into three categories of effect (modest, moderate, and strong) based on published odds ratios (supplementary table 4).

Retrospective estimation of overall pathogenic variant detection rates among EOAD patients

To compare APP, PSEN1 and PSEN2 LP/P variant detection rates in EOAD overall with published data, and because most published LP/P variant detection rates were obtained from smaller series, we estimated the LP/P variant detection rates among EOAD patients since the beginning of genetic screening in France in our national center. Overall, we consider that we have prospectively or retrospectively sequenced all probands whose DNA has been sent to our center since the 1990s and until November 2022, among those with (i) a neuropathological diagnosis, (ii) a clinical diagnosis of AD with supporting AD pathophysiology or (iii) a typical AD diagnosis, in absence of biomarkers available.

Exome programs in a research setting were focused on individuals from European ancestry, to match with available data in terms of control individuals for case-control analyses, explaining why we restricted our estimates to this population.

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Results

Patient enrollment

Over 30 months, we included 700 patients from 40 French centers. Of them, 608 (87%) presented EOAD and 92 (13%) had LOAD (66<AOO<76 years); 422 (60.3%) were female and 278 (39.7%) were male. All but 5 patients had CSF biomarkers available and consistent with an AD pathophysiological process; these 5 patients showed positive amyloid PET scans. Among EOAD patients, 258 (42.4%) were sporadic or family history was unknown and 350 (57.6%) had a positive history, including 230 for whom the medical history of AD consisted of one or several late-onset cases and 120 with at least one relative with EOAD (Figure 3). Among the latter, 95 pedigrees showed that EOAD spanned at least two generations, thus suggesting possible autosomal dominant EOAD. Among LOAD patients, 61 (66.3%) had a positive family history, and 31 (33.7%) were sporadic cases or family history was unknown. There was no significant difference in the rates of positive family history between EOAD and LOAD cases. The percentage of males was 40% among EOAD patients and 36% among LOAD patients (Non Significant NS).

Variants in Mendelian genes

All results were delivered to the referring clinician after a median of 5.6 months (range: [0.8-11.1]); only 5 patients had a delay from inclusion to genetic report >10 months, for technical reasons).

Following clinical interpretation of an in silico gene panel of 28 genes associated with monogenic causes of dementia, we identified 21 patients (3%) harboring a likely pathogenic or pathogenic (LP/P) variant considered as the cause of the disease, all in EOAD patients: 14 in PSEN1, 4 in APP, 2 in PSEN2 (Table 1) and one patient exhibited a pathogenic variant NM_007375. In addition, four LP/P variants in Mendelian genes were considered, all in EOAD patients, and were interpreted as putatively contributing to the phenotype but not necessarily causing AD (supplementary information, supplementary table 1). Overall, 7 class-3 variants (uncertain significance) were returned to patients. One of them (in PSEN1) could later be reclassified as benign following mRNA assessment, and another one (in MAPT) led to question the AD diagnosis (supplementary table 2).

Rates of LP/P variants based on pedigree structure

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All 21 LP/P variants considered as causing dementia were found in EOAD patients (3.5%), including 12/120 patients with a family history of EOAD (familial-early, 10%), which represented 11/95 (11.6%) among pedigrees with EOAD spanning at least 2 generations (Figure 3).

In EOAD patients with a family history of LOAD (familial-late) or unknown AOO in the affected relative, only 3 LP/P variants were detected, in addition to the above-mentioned TARDBP pathogenic variant. This includes a patient carrying NM_000447.3:c.850A>G p.(Arg284Gly) variant in PSEN2, with a history of LOAD in the father (DNA not available, death at 92 years of age) and one patient carrying NM_000021.4:c.346A>G p.(Thr116Ala) variant in PSEN1, with uncles and aunts affected by LOAD and living unaffected parents (DNA NA, aged 88 and 87 respectively at last visit of the proband). As both variants were expected to be fully penetrant, we hypothesize that these variants may be unrelated to the family history and could eventually be de novo variants (DNV), although we could not demonstrate it. The third case had a family history of AD with unknown AOOs in the mother and maternal uncles and aunts; his father was unknown, thus causing a censoring effect. He carried a NM_000021.4:c.806G>A p.(Arg269His) variant in PSEN1, already associated with some diversity of AOO, including ages > 65 years. Overall, no direct link between the LP/P variants and family history could be established in these three EOAD cases with a family history of LOAD.

Among sporadic EOAD patients, 5/258 (1.9%) showed a LP/P variant, including one DNV in the APP gene in a patient with an AOO of 42 years. The other four patients had AOOs ranging from 52 to 58 (Table 1). All four variants were either novel (NM_000021.

Rates of risk factors

Among patients without a monogenic cause of dementia (n=679), 154 (22.7%) were APOE-ε4 homozygous and 278 (40.1%) were heterozygous. Eighty-three patients (12.2%) carried at least one rare risk factor, including 5 probands carrying 2 of them (0.7%). Of these 83 patients, 14 (16.8%) were also APOE-ε4 homozygous and 34 (4.2%) were heterozygous, thus accumulating at least two risk factors and 12/83 were LOAD patients. Overall, we found at least one risk factor in 470/679 (69.2%) patients. Patients with at least one risk factor were enriched in the LOAD group (93%, as compared to 84% among EOAD patients, p=0.0009) and this was mainly due to the APOE4 allele which was present in 77% of LOAD patients and 61% of EOAD patients (p=0.0004; no significant difference in proportions J o u r n a l P r e -p r o o f of rare risk variants between EOAD and LOAD patients). Patients with at least one risk factor were more susceptible to show a positive family history (64% compared to 44%, p=1.4 10-6) and there was no significant difference in sex ratios.

The gene showing the largest number of risk variants was TREM2, with 11 NM_018965.4c.185G>A p.(Arg62His) (R62H) and 18 NM_018965.4:c.140G>A p.(Arg47His) (R47H) patients carrying this variant (supplementary table 3). One of them was homozygous for the TREM2 R47H variant and he also carried a missense variant of uncertain significance in SORL1, NM_003105.6:c.418G>A p.(Asp140Asn) (not reported). ABCA7 was the second most frequent gene, with 25 patients carrying at least one ABCA7 truncating variant. One patient harbored 2 truncating ABCA7 variants (AOO 65 years, family history of LOAD), but we could not determine if variants were in cis or in trans. The most recurrent ABCA7 truncating variant (n=8) was the splice region NM_019112.4:c.5570+5G>C variant, with a demonstrated LOF effect [START_REF] Steinberg | Loss-of-function variants in ABCA7 confer risk of Alzheimer's disease[END_REF] . The second most recurrent ABCA7 variant, NM_019112.4:c.2126_2132del p.(Glu709Alafs*86), was found in 5 patients and previously reported in diverse ethnicities [START_REF] Steinberg | Loss-of-function variants in ABCA7 confer risk of Alzheimer's disease[END_REF][START_REF] Guennec | ABCA7 rare variants and Alzheimer disease risk[END_REF][START_REF] Cuyvers | Mutations in ABCA7 in a Belgian cohort of Alzheimer's disease patients: a targeted resequencing study[END_REF] . The third gene in frequency was ATP8B4, with 20 patients being heterozygous for the NM_024837.4:c.1183G>A p.(Gly395Ser) (G395S) variant with a modest effect. Nine patients carried a SORL1 truncating variant, including one patient also harboring an ABCA7 truncating variant and an APOE-ε3/ε4 genotype; this patient had an AOO of 65 years and had a family history of EOAD. Except one doubleton, all SORL1 truncating variants were singletons.

Three patients carried a variant considered as a risk factor in ABCA1, including one truncating variant and two missense variants.

In addition to the already-mentioned patients, two carried multiple rare risk factors: one patient with a TREM2 R47H variant and an ATP8B4 G395S variant, in addition to an APOE-ε3/ε4 genotype (AOO 61, family history of EOAD) and one patient with an ABCA7 truncating variant and an ATP8B4 G395S variant, in addition to an APOE-ε3/ε4 genotype (AOO 62, sporadic case). Overall, 5 patients harbored two rare risk factors, if counting the TREM2 R47H homozygote double, and 3 of them carried these variants in an APOE-ε3/ε4 context (Supplementary Figure ).

When focusing on patients with a pedigree suggestive of autosomal dominant EOAD (EOAD spanning two generations), 74/84 (88.1%) carried at least one risk factor, and 12/84 (14.3%) carried at least one rare risk factor, suggesting that EOAD may be oligogenic in these patients too.

Retrospective estimation of overall pathogenic variant detection rates among EOAD patients

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To expand these prospective findings and with the aim to contribute to the discussion on genetic screening recommendations in AD, we performed a retrospective analysis of all EOAD unrelated probands from European descent assessed by exome or Sanger sequencing (or both) since the discovery of the APP, PSEN1 and PSEN2 genes, until November 2022, in our national reference center. Of 2,069 patients, 255 exhibited a LP/P variant, thus the overall LP/P variant detection rate is 12.3% (95% confidence interval [10.9-13.7]).

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Discussion

In this prospective study, we assessed how a large genetic screen in AD patients may lead to reporting clinically-relevant genetic variants to patients, including Mendelian dementia genes and risk factors, in a clinical setting. There was no major issue in implementing exome sequencing for this rather large series, on a nationwide scale, demonstrating that it is possible and meaningful to propose exome sequencing to basically any patient with EOAD, despite the relatively high frequency of AD as compared to other causes of early-onset dementia.

Clinical utility may be divided into two main outputs, i.e. LP/P variant detection rates in Mendelian genes and risk factor detection rates.

We previously assessed LP/P variant detection rates in APP, PSEN1 and PSEN2 genes in retrospective series [START_REF] Nicolas | Screening of dementia genes by whole-exome sequencing in early-onset Alzheimer disease: input and lessons[END_REF][START_REF] Lacour | Causative Mutations and Genetic Risk Factors in Sporadic Early Onset Alzheimer's Disease Before 51 Years[END_REF][START_REF] Wallon | The French series of autosomal dominant early onset Alzheimer's disease cases: mutation spectrum and cerebrospinal fluid biomarkers[END_REF] . In a first series, we focused on EOAD patients with a positive family history of EOAD, so that cases spanned at least 2 generations. The LP/P variant detection rate was 77% [START_REF] Wallon | The French series of autosomal dominant early onset Alzheimer's disease cases: mutation spectrum and cerebrospinal fluid biomarkers[END_REF] . Here, after restriction to the same definition of pedigrees, we reached an 11.6% variant detection rate, despite the use of a more sensitive technology. Several hypotheses may explain this difference. First, during the inclusion period of the ECASCAD study, our center received samples from individuals with EOAD, not included in the ECASCAD study because a LP/P variant was already known in their family, whether or not the referring clinician was aware of this, the link with a known family was made possible thanks to the family database of our center, acting as a unique center for EOAD genetics since the 1990s. Overall, 15 patients from 15 different families were assessed by targeted sequencing of the familial pathogenic variant during the same inclusion period as the ECASCAD study. If these variants had not been known from the laboratory, these patients would have been considered as probands and would have been included in the ECASCAD study, thus reaching a 24% detection rate in the category of EOAD spanning two generations, still somewhat away from the expected 77%. Second, since more than two decades, presymptomatic testing can be requested, so that some of the asymptomatic heterozygotes for LP/P variants may have become symptomatic in the meantime, obviously not requesting another genetic test after symptoms onset and not adding up to the above-mentioned number of symptomatic relatives. Third, the recruitment of EOAD probands has evolved since our 2012 report, with an average AOO of EOAD patients sent for genetic testing of 54.3 years before 2012 and 57.3 years here (p<0.001) and a more stringent selection of cases of sequencing prior to the 2000s (e.g. requirement of a family history positive for EOAD and spanning 3 generations rather than 2 at the beginning of the lab activity, or a bias towards a lower proportion of APOE ε4-positive probands). Thus, despite unbiased prospective inclusion of cases here, the prospective variant detection rate seems to deeply depend on the history of genetics screening in the country. We hypothesize that the majority of families of French ancestry with fully penetrant variants may already be known, and that families J o u r n a l P r e -p r o o f with non-French ancestry, atypical pedigrees and less penetrant variants may still be a source of novel families with a LP/P variant, to which should be added de novo variants.

We previously established national recommendations for APP, PSEN1 and PSEN2 screening, which were: diagnosis of EOAD in the proband and presence of a family history of EOAD (whatever the generation) or the AOO in the proband is <51 years. In the latter category of patients, we previously detected a 12.3% LP/P variant detection rate in a retrospective study [START_REF] Lacour | Causative Mutations and Genetic Risk Factors in Sporadic Early Onset Alzheimer's Disease Before 51 Years[END_REF] , most of them were de novo variants after assessing parental DNA, when available. Here, only 1/31 (3.2%) patient fulfilling these criteria was heterozygous for an APP pathogenic variant, which was also confirmed to be de novo.

In our previous national recommendations, patients with an AOO between 51 and 65 and a negative family history or a family history of LOAD were not prioritized for genetic screening in a diagnostic setting. After retrospective sequencing of such cases, we previously identified 1.2-2.2% LP/P variant detection rates [START_REF] Nicolas | Screening of dementia genes by whole-exome sequencing in early-onset Alzheimer disease: input and lessons[END_REF] . Here, the rate of LP/P variants in patients with sporadic EOAD and AOO between 51 and 65 or patients with EOAD and a family history of LOAD was 1.5%, which is thus consistent with our previous report.

To better compare our LP/P variant detection rate in APP, PSEN1 and PSEN2 with the literature, we gathered data from our national center for all EOAD probands with available sequencing data since the discovery of these genes. We found a LP/P variant detection rate of 12.3% [10.9-13.7]. Although this seems consistent with previous reports in other countries, [START_REF] Giau | PSEN1, and PSEN2 Mutations in Asian Patients with Early-Onset Alzheimer Disease[END_REF][START_REF] Jia | PSEN1, PSEN2, and APP mutations in 404 Chinese pedigrees with familial Alzheimer's disease. Alzheimer's & dementia : the journal of the Alzheimer[END_REF] this can still be slightly overestimated because of hard selection of cases at the beginning of the studies in the 1990s.

In some common disorders, in which monogenic causes can be hidden in a minority of patients (e.g. cancers, cardiovascular diseases), prioritization algorithms based on a priori probabilities of finding a pathogenic variant are often used. For actionable diseases, wider indications of genetic screening are proposed, up to universal reflexive testing, not to miss therapeutic opportunities [START_REF] Murray | Addressing the routine failure to clinically identify monogenic cases of common disease[END_REF] .

Although AD is not highly actionable, it appears as critical for genetic counseling, not to miss a LP/P variant in an EOAD proband requesting a genetic analysis. In addition, in anticipation to putative preventive treatments in presymptomatic individuals with monogenic AD variants [START_REF] Salloway | A trial of gantenerumab or solanezumab in dominantly inherited Alzheimer's disease[END_REF] , which could even be extended to oligogenic combinations of moderate-to-strong risk factors someday, and following promising clinical trials based on the use of recently proven effective molecules as disease-modifying treatments [START_REF] The | Lecanemab for Alzheimer's disease: tempering hype and hope[END_REF][START_REF] Mcdade | Lecanemab in patients with early Alzheimer's disease: detailed results on biomarker, cognitive, and clinical effects from the randomized and open-label extension of the phase 2 proof-of-concept study[END_REF][START_REF] Van Dyck | Lecanemab in Early Alzheimer's Disease[END_REF] , variant detection rates should no longer be the main argument for proposing a genetic test in the context of EOAD. In addition, it seems, from the current study, that there is no longer a big advantage, in terms of variants detection rate, to prioritize genetic screening based on AOO and pedigree structure, among EOAD patients. Indeed, after more than 25 years of genetics screening of EOAD patients in France, the lowest LP/P variant detection rate among novel EOAD patients was 1.7% and the highest, 11.6%, depending on AOO and pedigree structures. Thus, we consider that all EOAD J o u r n a l P r e -p r o o f patients should be offered a genetic screen, provided that the clinician requesting the analysis is trained in such a prescription and results delivery.

Although the inclusion of LOAD patients was too limited, preventing us from generalizing our results, we did not identify any LP/P variant in this AOO category. Some APP, PSEN1 or PSEN2 variants have already been identified in patients with an AOO > 65 years, with extremely low variant detection rates. For example, in the Alzheimer Disease European Sequencing (ADES) study encompassing patients from Europe and patients from the USA (Alzheimer Disease Sequencing Project, ADSP), 0.3% of the LOAD patients carried a LP/P variant in the discovery stage of this study [START_REF] Holstege | Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease[END_REF] . Some of these variants were known to be associated with reduced penetrance and later AOO. Thus, genetic screen in LOAD patients should rely on a case-by-case basis and sequencing is not widely recommended.

Interestingly, the rate of LP/P variants in genes causing differential diagnoses was extremely low, suggesting that using IWG-2 criteria drastically reduces the likelihood of non-AD diagnoses. In addition, the only patient with a redirection of the diagnosis towards FTLD was questionable because of borderline evidence of amyloidopathy and uncommon phenotype for AD. Besides, another patient, with a class-3 MAPT variant, showed normal Aβ42 levels following a second lumbar puncture (supplementary table 3). Although a final diagnosis of FTLD cannot be confirmed in the latter patient based on genetic arguments, the diagnosis is still uncertain and not in favor of AD anymore. This study therefore underlines the importance on relying on etiological biomarkers in atypical and typical presentations.

The second aim of our study was to assess the rate of clinically relevant genetic risk factors.

Although the clinical impact may appear as limited for patients, because risk factors cannot be used for genetic counseling, variants with OR>1.5, and most likely variants >2 and even more if >5 may still be clinically meaningful. Here, we developed a framework for the classification of AD risk factors in a clinical setting and identified that 69.2% of the probands without a monogenic cause exhibited at least one definite AD risk factor. This included 12.2% of patients carrying so-called rare risk factors, thus showing that such risk factors affect a significant proportion of patients and may thus be considered as rather common in EOAD and even among young LOAD patients. Such results suggest oligogenic inheritance in some patients, especially in case of identification of multiple risk factors, and this includes families with a pedigree suggesting autosomal dominant inheritance. We suggest that identifying one or multiple moderate to strong risk factors in EOAD patients may help families understand the etiology of the disease, although they are not sufficient to fully explain AD occurrence in the cases. Patients with early-onset dementia and their caregivers or relatives often wonder about J o u r n a l P r e -p r o o f the etiology of such an elderly-associated disease occurring at a young age. Delivering etiological factors, even if not actionable, may thus be useful. In addition, risk variants bring some pieces of evidence that AD may eventually be non-monogenic, combined with a negative screen of Mendelian genes, thus providing some reassurance to families regarding recurrence risk in relatives, not reaching 50% for first-degree relatives. In addition, if the future of AD management includes prevention strategies, better understanding the role of such moderate-to-strong risk factors appears to be useful.

Of note, we used stringent criteria for variant selection, so that we returned only definite risk factors to patients. A number of variants of uncertain significance were identified but not reported. Indeed, if gene-based tests provided clear evidence of an AD-risk association of burdens of rare, predicted damaging missense and truncating variants in the selected genes, coming back to the individual and the variant levels should rely on ACMG-AMP-like arguments, which did not allow most of the missense variants to reach sufficient evidence to be reported to patients.

We report here the results of a prospective study until the delivery of the result. Of note, some of the variants may be reclassified in the future (more specifically, variants of uncertain significance, as for example one of the PSEN1 variants later reclassified as likely benign here), and some novel genes may be considered, at least among risk factor genes. Thus, producing and interpreting such exome sequencing data should be accompanied by a service of results reinterpretation, especially for variants of uncertain significance, but also for negative results, upon request of the patient/family and referring physician.

One weakness of our study is the limitation to individuals from European ancestry. Although we regret that limitation, providing the information on risk variants requires (i) the replication of the results of association studies in the same ethnicities as patient and (ii) estimates of odds ratios in large series with inclusion criteria as close as possible to those used in the clinic. Unfortunately, association studies have mainly been performed on individuals from European ancestry, especially the latter large exome sequencing study that unveiled the role of rare variants in ABCA1 and ATP8B4 [START_REF] Holstege | Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease[END_REF] . For the latter two genes, there is indeed only one study, with results replicated within this study, but all datasets are based on individuals with European ancestries. For other genes, such as ABCA7 or TREM2, pieces of information are available in African-Americans, for example, as recently reviewed [START_REF] Logue | Genetics of Alzheimer's Disease in the African American Population[END_REF] . However, most results on rare variants remain not significant after multiple testing correction, probably due to insufficient power, or odds ratios are not computed. The increased number and size of genetic studies in other ethnicities will allow the reduction of such limitations in the future. However, only risk variants seem to be affected by such limitations. There is no reason that monogenic dementia variant detection rates would be affected, and our overall results are consistent with the literature in diverse ethnicities [START_REF] Giau | PSEN1, and PSEN2 Mutations in Asian Patients with Early-Onset Alzheimer Disease[END_REF][START_REF] Jia | PSEN1, PSEN2, and APP mutations in 404 Chinese pedigrees with familial Alzheimer's disease. Alzheimer's & dementia : the journal of the Alzheimer[END_REF] . Thus, our recommendations on genetic screening of monogenic dementia genes in AD may well apply to any ethnicity.

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Another limitation is that we did not consider risk factors with odds ratios below 1.5, most of them having been identified in genome-wide association studies based on DNA chips [START_REF] Bellenguez | New insights into the genetic etiology of Alzheimer's disease and related dementias[END_REF] . Such variants can be combined into polygenic scores, which could also provide part of the etiological explanation.

However, beyond the fact that most of these variants are not in coding exons -and thus not detectable by exome sequencing -polygenic scores deeply rely on the large effect of the APOE4 allele, which was taken into account in our study. The combined effect of non-APOE related variants remains modest. In addition, we can speculate that information on polygenic scores may be more difficult to understand for patients and families.

Here, we used exome sequencing as a cost-effective tool to (i) accurately detect variants in the selected gene list and (ii) allow the reinterpretation of results in light of the discovery of novel genes in the future, without the need to resequence. Sequencing only a gene panel would be expected to provide similar results, with a limitation for future reanalyses, while proposing genome sequencing remains more costly, with very limited gains in terms of variant detection. Gene panels might be seen as more powerful for CNV detection than exome sequencing, but we previously showed similar performances when CNVs affect at least two targets (i.e. two exons), as the design of baits for exon capture in panels and exomes is usually very similar, with the exception of panels that capture introns, although such a design is not common [START_REF] Quenez | Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation[END_REF] . Some smaller CNVs, i.e. single-exon CNVs, may be missed by bioinformatics tools when working from exon capture, but in a similar manner between panel and exome sequencing. For these reasons, we do not recommend using preferentially gene panel or exome sequencing or genome sequencing, as the availability of these techniques depends on the organization of genetic testing in a given country and results are expected to be comparable.

In conclusion, after decades of genetic screening of autosomal dominant genes in EOAD patients, we recommend to analyze dementia genes by sequencing all probands with EOAD, whatever the family history. The clinical utility of the identification of LP/P variants in Mendelian genes is clear, given the consequences for genetic counseling, and it may be even higher if preventive treatments become available in the future. The clinical utility of moderate-to-strong risk factors relies on the need for some patients and families to receive some (partial) answers on disease etiology and on reducing the likelihood of a missed LP/P variant in a Mendelian gene. The clinical impact of returning risk factors according to patients and clinicians should be assessed specifically in anticipation of putative clinical use.

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  3:c.1144G>C p.(Ala382Pro) in an AD differential diagnosis gene, TARDBP. Re-examining clinical information allowed us to reconsider the diagnosis towards Fronto-Temporal Lobar Degeneration (FTLD) for the latter patient, who presented behavioral symptoms and borderline amyloidopathy based on CSF biomarkers (supplementary information).

  4:c.289G>A p.(Val97Met) in PSEN1 and NM_000447.3:c.365C>T p.(Thr122Met) in PSEN2) or described in only one patient before (NM_000021.4:c.800C>T p.(Pro267Leu) in PSEN1, in an autosomal dominant EOAD family proband 25 , and NM_000021.4:c.1309A>G p.(Ile437Val) in PSEN1, in a sporadic patient 9 ), thus with insufficient information on penetrance.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Study procedures Figure 2. Framework for the interpretation and classification of risk factors in Alzheimer disease Truncating variants are defined as nonsense, canonical splice site or frameshift variants predicted to trigger nonsense-mediated decay (not concerning the last coding exon or 50 last bp of the penultimate exon) and not affecting small (<100bp) in-frame exons for splice site variants or single exon deletions. Strength of effect is classified based on OR and appears in bold: modest if 1.5<OR<2, moderate if 2<OR<5 and strong if OR>5 TREM2 R47H: NM_018965.4:c.140G>A p.(Arg47His) TREM2 R62H: NM_018965.4c.185G>A p.(Arg62His) ATP8B4 G395S: NM_024837.3:c.1183G>A p.(Gly395Ser)

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Acknowledgements

We thank all patients and families for participating to the study. Collaboration CEA-DRF-Jacob-CNRGH-CHU de Rouen.

Funding

This study was funded by Fondation Alzheimer (ECASCAD study). This work did benefit from support of the France Génomique National infrastructure, funded as part of the «Investissements d'Avenir» program managed by the Agence Nationale pour la Recherche (contract ANR-10-INBS-09).

Data availability

All variants of clinical interest are available in tables and supplementary tables.

Author contributions

Conceptualization

Ethics declaration

This study has been approved by the ethics committee CPP-Ouest III (notification 2018-A02359-46).

Conflicts of interest