Contribution of homozygous and compound heterozygous missense mutations in VWA2 to Alzheimer's disease

University of Groningen

Contribution of homozygous and compound heterozygous missense mutations in VWA2 to

Alzheimer's disease

BELNEU Consortium; Hoogmartens, Julie; Hens, Elisabeth; Engelborghs, Sebastiaan;

Vandenberghe, Rik; De Deyn, Peter-P; Cacace, Rita; Van Broeckhoven, Christine

Published in:

Neurobiology of Aging

DOI:

10.1016/j.neurobiolaging.2020.09.009

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BELNEU Consortium, Hoogmartens, J., Hens, E., Engelborghs, S., Vandenberghe, R., De Deyn, P-P.,

Cacace, R., & Van Broeckhoven, C. (2021). Contribution of homozygous and compound heterozygous

missense mutations in VWA2 to Alzheimer's disease. Neurobiology of Aging, 99, 100.e17-100.e23.

https://doi.org/10.1016/j.neurobiolaging.2020.09.009

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Contribution of homozygous and compound heterozygous

missense mutations in VWA2 to Alzheimer

’s disease

Julie Hoogmartens

, Elisabeth Hens

, Sebastiaan Engelborghs

,

Rik Vandenberghe

, Peter-P. De Deyn

, Rita Cacace

_,

Christine Van Broeckhoven

_{, on behalf of the BELNEU Consortium}

a_{Neurodegenerative Brain Diseases Group, VIB Center for Molecular Neurology, Antwerp, Belgium} b_{Institute Born-Bunge, Antwerp, Belgium}

c_{Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium}

d_{Department of Neurology and Memory Clinic, Hospital Network Antwerp, Middelheim and Hoge Beuken, Antwerp, Belgium} e_{Department of Neurology, University Hospital Antwerp, Edegem, Belgium}

f_{Department of Neurology, University Hospital Brussel and Center for Neurosciences, Free University Brussels, Brussels, Belgium} g_{Department of Neurology, University Hospitals Leuven and Department of Neurosciences, KU Leuven, Belgium}

a r t i c l e i n f o

Received 1 June 2020

Received in revised form 2 September 2020 Accepted 4 September 2020

Available online 12 September 2020 Keywords:

Alzheimer’s disease

Von Willebrand factor A domain containing 2 gene

VWA2

Homozygous and compound heterozygous missense mutations

a b s t r a c t

Alzheimer’s disease is the most frequent diagnosis of neurodegenerative dementia with early (
65 years) and late (>65 years) onset ages in familial and sporadic patients. Causal mutations in 3 autosomal dominant Alzheimer genes, i.e. amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2), explain only 5%e10% of early-onset patients leaving the majority of patients genetically unre-solved. To discover potential missing genetics, we used whole genome sequencing data of 17 early-onset patients with well-documented clinical diagnosis of Alzheimer’s disease. In the discovery group, the mean onset age was 55.71 6.83 years (range 37e65). Six patients had a brain autopsy and neuropa-thology confirmed Alzheimer’s disease. Analysis of the genetic data identified in one patient a homo-zygous p.V366M missense mutation in the Von Willebrand factor A domain containing 2 gene (VWA2). Resequencing of the VWA2 coding region in an Alzheimer's disease patient cohort from Flanders-Belgium (n¼ 1148), including 152 early and 996 late onset patients, identified additional homozygous and compound heterozygous missense mutations in 1 early and 3 late-onset patients. Allele-sharing analysis identified common haplotypes among the compound heterozygous VWA2 mutation carriers, suggesting shared ancestors. Overall, we identified 5 patient carriers of homozygous or compound heterozygous missense mutations (5/1165; 0.43 %), 2 in early (2/169; 1.18 %) and 3 in late-onset (3/996; 0.30 %) patients. The frequencies of the homozygous and compound heterozygous missense mutations in patients are higher than expected from the frequencies calculated based on their combined single alleles. None of the homozygous/compound heterozygous missense mutation carriers had a family history of autosomal dominant Alzheimer’s disease. Our findings suggest that homozygous and compound heterozygous missense mutations in VWA2 might contribute to the risk of Alzheimer’s disease in sporadic patients.

Ó 2020 Elsevier Inc. All rights reserved.

1. Introduction

Progressive loss of memory and disturbances of cognitive

functions such as word-finding, spatial cognition and

problem-solving clinically characterize Alzheimer’s disease (AD) (Cacace

et al., 2016). Extracellular accumulated amyloid-

b

b

and intracellular formation of neurofibrillary tangles of

hyper-phosphorylated tau proteins are the neuropathological hallmarks of AD (McKhann et al., 2011). These neuropathological characteristics are accompanied by gliosis and loss of neurons and synapses

Funding: The research was in part supported by the Flemish Government initiated Methusalem excellence program, the Flanders Impulse Program on Net-works for Dementia Research (VIND) and the Research Foundation Flanders (FWO); Belgium. R.C. received a postdoctoral grant from the FWO.

* Corresponding author at: VIB Center for Molecular Neurology, University of Antwerp - CDE, Universiteitsplein 1, 2610 Antwerp, Belgium. Tel.:þ32 3 265 1039; fax:þ323 625 84 10.

** Corresponding author at: VIB Center for Molecular Neurology, University of Antwerp - CDE, Universiteitsplein 1, 2610 Antwerp, Belgium. Tel:þ32 3 265 1101; fax:þ323 625 84 10.

E-mail addresses: [email protected] (R. Cacace), christine.

[email protected](C. Van Broeckhoven).

Contents lists available atScienceDirect

Neurobiology of Aging

j o u rn a l h o m e p a g e : w w w . e l s e v ie r . c o m / l o c a t e / n e u a g i n g

0197-4580/$e see front matter Ó 2020 Elsevier Inc. All rights reserved.

(Cacace et al., 2016; Ringman et al., 2014). Among all dementia subtypes, AD is the most frequent and is affecting up to 75 % of dementia patients (Cacace et al., 2016;Koedam et al., 2010). Aging is the most prominent biological risk factor for developing AD at late age, and around 90 % of patients are diagnosed above 65 years (Cacace et al., 2016). In the early-onset AD (EOAD) patients, diag-nosed before 65 years, genetic risk is more prominent (Wingo et al.,

2012). Highly penetrant mutations in the amyloid precursor protein

(APP), presenilin 1 (PSEN1) or presenilin 2 (PSEN2), identi_{fied in}

extended early-onset families with autosomal dominant

trans-mission of AD, explain about 5%e10% of all EOAD patients (Cacace

et al., 2016;Cruts et al., 1998;van Duijn et al., 1991). The

apolipo-protein (APOE)ε4 allele is a major risk factor for AD at late age

(Cacace et al., 2016), increasing risk 3 times in heterozygous- and 15 times in homozygous carriers (Corder et al., 1993). At early age, risk

is also increased in homozygous carriers and heterozygousε4

car-riers with a positive family history of dementia (van Duijn et al.,

1994). Additionally, APOE alleles act as onset age modifiers in

autosomal dominant AD families segregating a known pathogenic mutation (Cacace et al., 2016). Family studies showed that the onset age of the patient was earlier in the presence of an 34 allele and later in the presence of an 32 allele, which is a protective allele (Cacace

et al., 2016; Sorbi et al., 1995). The use of large scaled genome-wide association studies (GWAS) and next generation sequencing

(NGS) datasets ultimately identified additional genes with variable

genetic contributions to AD risk (e.g., [Cacace et al., 2019;Cuyvers and Sleegers, 2016; Guerreiro et al., 2012; Jansen et al., 2019;

Zhou et al., 2019]). Together, the causal and risk genes explain still a small fraction of AD patients, leaving the majority of familial and

sporadic patients genetically unresolved (Brouwers et al., 2008;

Jarmolowicz et al., 2015).

The identification of highly penetrant mutations in autosomal

dominant families led to the common perception that EOAD is solely caused by dominant alleles (Wingo et al., 2012). However, the

observation that in different studies only 35%e60% of EOAD

pa-tients have a clear positive family history does not support this perception (Cacace et al., 2016). Furthermore, only 10%e15% of the EOAD families show a clear autosomal dominant transmission (Campion et al., 1999;Jarmolowicz et al., 2015). These observations suggest that recessive inheritance of AD might contribute to the

missing genetic etiology (Wingo et al., 2012). Consequently, we

investigated in unexplained familial and sporadic EOAD patients, whether a recessive pattern of inheritance could contribute to their disease. Initially, we selected 17 EOAD patients with well-documented clinical or pathological AD diagnosis for

whole-genome sequencing (WGS) analysis and identified a homozygous

missense mutation in the Von Willebrand factor A domain con-taining 2 gene (VWA2). Genetic screening of VWA2 in AD patient

cohorts from Flanders-Belgium, identified additional homozygous

and compound heterozygous mutations (all missense mutations) that may mimic autosomal recessive inheritance.

2. Materials and methods 2.1. Patient and control cohorts

The AD patients were sampled in neurology expertise centers at university and general hospitals in the Flanders region of Belgium. The patients received a clinical AD diagnosis based on the diag-nostic criteria of the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the AD and Related Disorders Association (ADRDA) or the National Institute on

Aging-Alzheimer’s Association (NIA-AA) (Hyman et al., 2012;

McKhann et al., 1984,2011). The AD patient cohort from

Flanders-Belgium (n¼ 1165) included 169 EOAD patients (mean onset of

57.93 6.33 years [range 29e65]) and 996 late onset (LO) AD

pa-tients (mean onset of 77.60 6.32 years [range 66e95]) (Table S1).

Neuropathology at autopsy confirmed the clinical diagnosis of AD in

25 EOAD and 61 LOAD patients (Table S1). Sequencing of APP, PSEN1

and PSEN2 in the AD patient cohort identified in APP one pathogenic

mutation in an EOAD patient (0.085 %) and in PSEN1 11 pathogenic

mutations in 8 EOAD and 3 LOAD patients (0.944%) (Perrone et al.,

2020) (Table S2). Overall, we identified known pathogenic muta-tions in 1.030 % (12/1165) of the AD patients included in the cohort (Perrone et al., 2020). The cohort of control persons from the Flanders-Belgium region, included 759 age-matched individuals

with a mean age at inclusion (AAI) of 74.70 6.56 years (range

65e100), with a Mini-Mental State Examination (MMSE) or

Mon-treal Cognitive Assessment (MoCA) score26 and without a family

history of AD (Folstein et al., 1975; Nasreddine et al., 2005)

(Table S1). Genomic DNA (gDNA) was available of all participants, extracted from whole blood using standard laboratory procedures. 2.2. Ethical assurance

All participants or their legal representative signed an informed consent for the participation in clinical, neuropathological and ge-netic research. The ethical committee of the University Hospital of Antwerp and the University of Antwerp (Antwerp, Belgium) approved the clinical, neuropathological and genetics study protocols.

2.3. Genetic screenings

Complete Genomics, BGI, performed short reads paired end WGS (combinatorial probe-anchor ligation (cPAL) sequencing chemistry) (Drmanac et al., 2010). We achieved the genetic profiling

by means of massive parallel resequencing (Goossens et al., 2009)

or Sanger sequencing. For the multiplex amplification of 96

ho-mozygous mutations and targeted resequencing of all 13 coding exons of VWA2 (NM_001272046.2), we designed amplicon target

amplification assays. For primer design, we used the mPCR software

in which the target region size was set at 500 nucleotides. Using

multiplex polymerase chain reactions (PCR), we amplified target

regions followed by purification of the equimolar-pooled amplicons

using Agencourt AMPureXP beads (Beckman Coulter, CA, USA). We incorporated unique genetic barcodes (Illumina Nextera XT) in a universal PCR step prior to sample pooling. We sequenced the li-braries on a MiSeq platform, using the v2 reagent kit for variant genotyping and the v3 reagent kit for the full exonic resequencing of VWA2, with a paired-end read length of 250 (or 300) base pairs

(Illumina, USA). Identified variants were validated using Sanger

sequencing technology (BigDye Terminator Cycle Sequencing kit v3.1; analysis on an ABI 3730 DNA Analyzer, Applied Biosystems, Foster City, CA, USA).

2.4. Bioinformatics analysis

Complete Genomics, BGI, assembled raw reads to the reference genome (GRCh37, hg19) and performed sequencing alignment and variant calling for the WGS data. Annotation and analysis was

performed in-house using the GenomeComb pipeline (http://

genomecomb.sourceforge.net/) (Reumers et al., 2011). We per-formed the analysis of the resequencing experiments in-house. For alignment of raw reads to the reference genome (GRCh37, hg19), we

used the Burrow Wheeler Aligner (BWA) (Li and Durbin, 2009). For

variant calling, we used the Genome Analysis Toolkit (GATK) (DePristo et al., 2011;McKenna et al., 2010) and for variant anno-tation and downstream analysis, we used the in-house developed

GenomeComb annotation pipeline. Sequencing reads were visual-ized with Integrative Genomics Viewer (IGV) (Robinson et al., 2011). We searched in the WGS data for non-synonymous homozygous variants in the coding region (CDS) or conserved splice sites affecting the protein sequence. Good quality variants were selected as described (Reumers et al., 2011). We considered the variants with low coverage (<15X) or variants located in segmental duplications, repeated sequences or homopolymer stretches as false positives. We limited our search to variants with a minor allele frequency

(MAF)
5% in the Genome Aggregation (GnomAD) database v2.1.1

(Lek et al., 2016). To further prioritize the homozygous variants, we

consulted the Genotype-Tissue Expression (GTEx) portal (www.

gtexportal.org) and the healthy exome (HEX) database (478

healthy control individuals aged 60 years; neurodegenerative

brain diseases (NBDs) excluded [https://www.alzforum.org/

exomes/hex]). For the prediction of deleteriousness of identified amino acid changes, we used Combined Annotation Dependent

Depletion (CADD) version 1.6 (score20 is considered deleterious).

We reported the rescaled (PHRED) score, which correlates with allelic diversity and variant pathogenicity (Kircher et al., 2014).

2.5. Allele sharing analysis of haplotypes

We selected 6 polymorphic short-tandem repeat (STR) markers flanking VWA2 with 3 STR markers on each side, D10S1265, D10S205, D10S543 and D10S1681, D10S1731, D10S221, covering a

region of 12,4 cM (Marsh_{field comprehensive Human genetic}

maps) (Broman et al., 1998). The STRs were PCR amplified using

fluorescently labeled primers and size separated based on Gen-escan 500Liz size standard (Applied Biosystems) on an ABI3730xl DNA analyzer (Applied Biosystems). We obtained the STR fragment lengths using the in-house developed Local Genotype Viewer

gen-otyping software (https://www.neuromicssupportfacility.be/). We

performed STR genotyping for the compound heterozygous VWA2 mutation carriers and their available family members.

3. Results

3.1. WGS in a discovery EOAD patient group identified a

homozygous mutation in VWA2

The discovery group included 17 EOAD patients (Table S1), of

whom 6 had a definitive diagnosis confirmed by neuropathological

analysis at autopsy. A positive family history was present in 6 pa-tients though without clear autosomal dominant transmission. Causal mutations in APP, PSEN1 and PSEN2, were absent in all pa-tients. APOE 34 risk alleles were present in 5 patients with 1 patient

carrying 2 4 alleles. WGS, performed for all 17 EOAD patients,3

identified 96 homozygous mutations leading to amino acid changes

in 87 genes. To narrow down the number, we genotyped all mu-tations in a subset of AD patients characterized by early onset ages (
70 years [n ¼ 260]) and a subset of control individuals (n ¼ 596) (Table S1). We continued with 10 homozygous mutations observed in 10 different genes since they were present in additional AD

pa-tients and absent in the control cohort (Table 1). From the 10

prioritized genes, VWA2, glialfibrillary acidic protein (GFAP) and T-Box Transcription Factor 15 (TBX15) included homozygous mutations with deleterious CADD scores that are absent in the HEX database. Moreover, all 3 genes are expressed in brain tissue (GTEx portal). Ultimately, we selected VWA2 as the strongest candidate gene based on the low MAF in GnomAD and the absence of additional

VWA2 homozygous mutations (MAF
5%) in the HEX database.

3.2. Resequencing of VWA2 in an AD patient cohort from Flanders-Belgium

We performed full exonic resequencing of VWA2 in 152 EOAD and 996 LOAD patients from the Flanders-Belgium region (Table S1). In the EOAD patient cohort, we identified an additional

carrier with the homozygous p.V366M mutation (DR1619) (Fig. 1A,

Table 2). In the LOAD patient cohort, we observed 1 carrier homo-zygous for the p.R69M mutation (DR1620) and 2 carriers with the compound heterozygous mutation p.V366M/p.L715F (DR199.1 and DR1625) (Fig. 1A,Table 2). We also found 20 single mutations with a

MAF
5% in the AD patient cohort, of which 12 were only present in

patients. (Fig. S1,Table S4).

3.3. Allele sharing analysis in compound heterozygous VWA2 mutation carriers

We used the 6 STR markers flanking VWA2 to analyze the

segregation of the p.V366M/p.L715F mutations of carrier DR199.1 to

one child (DR199.2) (Fig. 1B). The analysis showed that the

muta-tions are located in trans since the child inherited only mutation p.V366M. We also used the data of the 6 STR markers to examine if the 2 carriers of the same p.V366M/p.L715F mutations (DR199.1 and DR1625) could be genetically related. Both patients share a

5.44 cM haplotype (D10S543-D10S1731)flanking p.V366M on one

allele and a 4.54 cM haplotype flanking p.L715F

(D10S543-D10S1681) on the other allele (Fig. 1B).

3.4. Clinical phenotype of homozygous and compound heterozygous VWA2 mutation carriers

Overall, we identified 5 patient carriers with detailed clinical

information available. Four of the homozygous and compound heterozygous VWA2 mutation carriers had a diagnosis of probable

AD and one carrier had definite AD. All VWA2 patient carriers

pre-sented with various cognitive problems (Table S3). 4. Discussion

We identified 5 AD patients (5/1165, 0.43 %) with homozygous

or compound heterozygous mutations in VWA2, of which 2 EOAD patients (2/169; 1.18 %) and 3 LOAD patients (3/996; 0.30 %) (Table 2). Four carriers (4/5, 80 %) were clinically diagnosed with

Table 1

Genes with homozygous mutations affecting the protein sequence Chr Gene EOAD carriers DAAa _HEX databaseb MAF (%) GnomAD Brain expressionc CADD score 1 TBX15 3 p.M460R 0 4.6 Yes 25.4 3 ACAA1 3 p.H231Q 5 5.0 Yes 0.7 4 FGFBP2 2 p.R60H 1 3.9 Yes 11.1 5 YTHDC2 3 p.L953V 1 4.8 Yes 13.7 10 VWA2 2 p.V366M 0 1.3 Yes 23.8 10 TEX36 2 p.V181I 0 3.9 No 0.001

12 MYO1A 2 p.G662E 1 3.5 Yes 12.0 17 GFAP 3 p.D157N 0 1.8 Yes 24.5 17 RHBDF2 2 p.M591V 0 2.4 Yes 18.0 20 BPIFB1 3 p.T464S 0 3.6 No 4.26 Key: DAA, amino acid substitution; CADD, Combined Annotation Dependent Depletion version 1.6 (Kircher et al., 2014); Chr, chromosome; EOAD, early-onset Alzheimer’s disease; GnomAD v2.1.1, Genome Aggregation database Non_Finnish European v2.1.1. (Lek et al., 2016); HEX, Healthy Exome; MAF, minor allele frequency.

a _{According to NP_001258975.1.}

b _{Number of homozygous carriers in the HEX database (https://www.alzforum.}

org/exomes/hex).

probable AD and one carrier (1/5, 20 %) with de_{finite AD at autopsy.} The homozygous and compound heterozygous VWA2 mutation carriers did not have a family history of autosomal dominant AD

and were free from causal mutations in APP, PSEN1 and PSEN2. There is not a clear influence of the APOE34 allele though the late

onset age of DR1620 (89 years) might have been influenced by the

Fig. 1. Genetic data of homozygous and compound heterozygous VWA2 mutation carriers from Flanders-Belgium. (A) Linear representation of alleles in VWA2 identified as ho-mozygous or compound heterozygous mutations in AD patients (n¼ 1165) from Flanders-Belgium. Protein domains are based on data from the UniProtKB/Swiss-Prot database

(UniProt, 2019) and protein nomenclature according to NP_001258975.1. VWA2 encodes a 755aa long protein including 3 VWFA domains (VWFA1, aa51-aa222; VWFA2,

aa343-aa517; VWFA3, aa531-aa705) and 2 EGF-like domains (EGF1, aa296-aa333; EGF2, aa712-aa748). (B) Allele sharing analysis of haplotypes in compound heterozygous VWA2 mu-tation carriers and available family members (DR199.1, DR199.2, and DR1625). Open symbols represent unaffected (at-risk) relatives andfilled symbols represent AD patients. Age at onset of patients and age at inclusion of unaffected relatives are indicated below the symbol. Slash, deceased;þ, age at death; arrowhead, index patient. Abbreviations: AD, Alzheimer’s disease; VWFA, Von Willebrand factor A; EGF, epidermal growth factor; aa, amino acid.

Table 2

AD carriers of homozygous and compound heterozygous VWA2 mutations

Individual AAO APOE genotype DAAa _{CADD score MAF GnomAD (%) MAF patient} cohort (%), n¼ 1165 F in patientsb_(%) F expectedc_(%) DR1617 64 34 p.V366M 23.8 1.27 1.80 0.17 0.023 p.V366M 23.8 1.27 1.80 DR1619 60 44 p.V366M 23.8 1.27 1.80 0.17 0.023 p.V366M 23.8 1.27 1.80 DR1620 89 23 p.R69M 27.5 0.27 0.60 0.086 0.0024 p.R69M 27.5 0.27 0.60 DR199.1 74 33 p.V366M 23.8 1.27 1.80 0.17 0.020 p.L715F 21.2 1.36 1.24 DR1625 85 33 p.V366M 23.8 1.27 1.80 0.17 0.020 p.L715F 21.2 1.36 1.24

Key:DAA, amino acid substitution; AAO, age at onset; AD, Alzheimer’s disease; APOE, apolipoprotein; CADD, Combined Annotation Dependent Depletion - version 1.6 (Kircher

et al., 2014); F, frequency; GnomAD, Genome Aggregation Database Non_Finnish European - v2.1.1 (Lek et al., 2016); MAF, minor allele frequency.

a_{According to NP_001258975.1.}

b _{Frequency of the observed homozygous/compound heterozygous mutations in the AD patient cohort from Flanders-Belgium (n¼ 1165).}

c _{Expected frequency of the homozygous/compound heterozygous mutations, calculated according to the Hardy-Weinberg principle using the MAF of single alleles in the AD}

(n¼ 2330 alleles) and control cohorts (n ¼ 1518 alleles) (Table S4).

APOE32 allele (Farrer et al., 1997). The variability in onset age of the patient carriers (60e89 years) is possibly due to different effects of the combined single alleles on the functioning of VWA2. The allele

combinations we observed are rare (frequency<0.1 %) based on

their frequency calculated in the Flanders-Belgian AD cohort (Tables 2and S4). Allele-sharing analysis of the compound

het-erozygous carriers identified shared haplotypes flanking p.V366M

and p.L715F, which is indicative for the presence of common

an-cestors. Evolutionary conservation analysis (ConSurf-DB

[Goldenberg et al., 2009]) showed that the amino acid positions of the homozygous/compound heterozygous mutations in patients

are evolutionary conserved, which is also reflected by the high

CADD scores (version 1.6: 21.2e27.5). We also screened the

Flanders-Belgian control cohort and observed one carrier with 2 mutations, p.R69M/p.L715F (1/759; 0.13 %), with an allele combi-nation that was absent in the AD patient cohort and with unknown cis/trans configuration.

The exact biological function of VWA2 remains unknown. VWA2 is not expressed in whole blood or in transformed lymphocytes (GTEx portal), limiting follow-up studies in easily accessible patient-derived biomaterials. Additionally, we were not able to

perform protein expression analysis in brain tissue of the de_finite

AD carrier as there was no fresh frozen tissue available. VWA2 is an extracellular protein including an N-terminal Von Willebrand factor (VWF) A domain, followed by a cysteine rich sequence, an epidermal growth factor (EGF)-like domain and 2 more VWFA do-mains (Richardson et al., 2014). At the C-terminal, another EGF-like domain is present (Richardson et al., 2014) (Fig. 1A). The

homozy-gous/compound heterozygous mutations identified in the patient

cohort are located inside 3 different domains; VWFA1, VWFA2 and

the EGF-like domain near the C-terminus (Fig. 1A). Based on

ho-mology, similar domains are found in numerous proteins (Davis,

1990;Tuckwell, 1999). The Von Willebrand factor A domains are a family of approximately 200 aa sequences, found predominantly in extracellular matrix proteins (Tuckwell, 1999). Generally, proteins containing VWFA domains are involved in cell-cell, cell-matrix and matrix-matrix interactions, and therefore play a role in important

processes like immunity (Sengle et al., 2003). In addition, a

sequence of around 40 aa, found in EGF, is present in a large number

of membrane-bound- and extracellular proteins (Davis, 1990;

Wouters et al., 2005). Many of these proteins require calcium for their biological function, which is believed to be crucial for numerous protein-protein interactions (Rao et al., 1995). However, investigation of the function and structure of the VWA2 protein indicated that VWA2 includes 2 EGF-like domains that are most

likely of the non-calcium binding variant (Sengle et al., 2003).

Interestingly, known AD associated genes like Sortilin Related Re-ceptor 1 (SORL1) and Notch reRe-ceptor 3 (NOTCH3) also contain EGF-like domains (UniProtKB/Swiss-Prot database).

Early studies demonstrated the co-localization of VWA2 with

basement membranes of several tissues (Sengle et al., 2003). In

zebrafish and mice, VWA2 contributes to the Fraser complex,

which stabilize basement membranes (Richardson et al., 2014).

Additionally, VWA2 was found in the proteome of extracellular

exosomes (Gonzalez-Begne et al., 2009). Exosomes are linked to

AD pathology, but their role is still controversial (Malm et al.,

2016). They are shown to spread toxic a

b

hyper-phosphorylated tau between cells and are believed to contribute to neuronal loss by inducing apoptosis (Malm et al., 2016). Yet, a number of studies have determined that neuron-derived

exo-somes can facilitate rapid conformational changes of A

b

nontoxic fibrils which can be internalized by microglia for

degradation (Howitt and Hill, 2016). Therefore, further research

remains necessary to con_{firm and explain the presence of VWA2}

in brain-derived exosomes.

To conclude, our genetic investigation of VWA2 identified 5

pa-tients carrying homozygous or compound heterozygous mutations in VWA2 and one control person with 2 mutations, for whom cis/

trans configuration was indecisive. These results suggest an

enrichment of homozygous/compound heterozygous mutation carriers among AD patients versus controls from Flanders-Belgium.

Taking into account the low frequencies of the identified

homozy-gous/compound heterozygous mutations and the limited number of participants in our Flanders-Belgian cohorts, there is need for

additional studies to support ourfindings. To obtain an adequate

power of 80%, we calculated that the number of subjects needed is

around 4000 (w2000 patients, w2000 controls). Anyway, in this

study we provide preliminary data suggesting that homozygous and compound heterozygous VWA2 mutations might contribute to sporadic AD by mimicking autosomal recessive inheritance and

increasing the risk for AD. Functional profiling of the observed

mutations and the investigation of the exact biological function of VWA2 is required to comprehend its role in the etiology of AD.

Disclosure statement

The authors have no conflicts of interest.

CRediT authorship contribution statement

Julie Hoogmartens: Conceptualization, Investigation, Writing -original draft, Writing - review & editing, Visualization. Elisabeth Hens: Investigation, Writing - original draft, Visualization, Re-sources, Validation. Sebastiaan Engelborghs: Investigation, Vali-dation, Resources. Rik Vandenberghe: Investigation, ValiVali-dation, Resources. Peter-P. De Deyn: Investigation, Validation, Resources. Rita Cacace: Conceptualization, Writing - original draft, Visualiza-tion. Christine Van Broeckhoven: Conceptualization, Resources, Writing - original draft, Writing - review & editing, Visualization, Supervision, Funding acquisition. P. Cras: Resources. J. Goeman: Resources. R. Crols: Resources. J.L. De Bleecker: Resources. T. Van Langenhove: Resources. A. Sieben: Resources. B. Dermaut: Re-sources. O. Deryck: ReRe-sources. B. Bergmans: ReRe-sources. J. Versijpt: Resources.

Acknowledgements

The following members of the BELNEU consortium have contributed to this paper by including patients in the Flanders-Belgian cohort and clinical and pathological phenotyping of the patients, as well as follow-up of the patients and families: Patrick Cras (University Hospital Antwerp, Edegem); Johan Goeman, Roe-land Crols (Hospital Network Antwerp, Antwerp); Jan L. De Bleecker, Tim Van Langenhove, Anne Sieben, Bart Dermaut (Uni-versity Hospital Ghent, Ghent); Olivier Deryck, Bruno Bergmans (General Hospital Sint-Jan Brugge, Bruges); and Jan Versijpt (Uni-versity Hospital Brussels, Brussels).

The authors are also thankful for the support of the personnel of the VIB CMN Neuromics Support Facility, the DNA Screening Facility of the VIB NBD research group, and the NeuroBioBank of the Institute Born-Bunge.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at https://doi.org/10.1016/j.neurobiolaging.

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