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Exome sequencing in an Italian family with Alzheimer’s disease points to a role for seizure-related gene 6 (SEZ6) rare variant R615H

  • Lara Paracchini
  • Luca Beltrame
  • Lucia Boeri
  • Federica Fusco
  • Paolo Caffarra
  • Sergio Marchini
  • Diego Albani
  • Gianluigi Forloni
Open Access
Research
  • 173 Downloads

Abstract

Background

The typical familial form of Alzheimer’s disease (FAD) accounts for about 5% of total Alzheimer’s disease (AD) cases. Presenilins (PSEN1 and PSEN2) and amyloid-β (A4) precursor protein (APP) genes carry all reported FAD-linked mutations. However, other genetic loci may be involved in AD. For instance, seizure-related gene 6 (SEZ6) has been reported in brain development and psychiatric disorders and is differentially expressed in the cerebrospinal fluid of AD cases.

Methods

We describe a targeted exome sequencing analysis of a large Italian kindred with AD, negative for PSEN and APP variants, that indicated the SEZ6 heterozygous mutation R615H is associated with the pathology.

Results

We overexpressed R615H mutation in H4-SW cells, finding a reduction of amyloid peptide Aβ(1–42). Sez6 expression decreased with age in a mouse model of AD (3xTG-AD), but independently from transgene expression.

Conclusions

These results support a role of exome sequencing for disease-associated variant discovery and reinforce available data on SEZ6 in AD models.

Keywords

Alzheimer’s disease SEZ6 Exome sequencing Rare variants 

Background

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder whose onset is mostly sporadic [1]. The genetic background has a major role in AD, and DNA variants may contribute, ranging from predisposing risk factors (having from medium to large effect size, such as the ε4 allele of the APOE gene) [2] to full penetrant causal mutations in a few genes, namely presenilins (PSEN1 and PSEN2) and the amyloid-β (A4) precursor protein (APP) [3, 4]. PSEN1/2 and APP gene mutations have been linked to early-onset, autosomal dominant familial forms of Alzheimer’s disease (FAD) [5, 6]. Recently, large-scale whole-exome sequencing has found rare variants reported to contribute to AD risk, such as in the PLCG2, ABI3, and TREM2 genes [7]. These findings indicate the involvement in familiar forms of AD of variants belonging to genes other than PSEN1/2 and APP, which may have a causal or predisposing role, as recently reported for SORL1 gene [8].

We report an Italian family with several cases of AD (having an onset between 60 and 70 years) negative for PSEN1/2 or APP mutations and whose available affected members were found to bear SEZ6 gene rare missense variant R615H. We describe the genetic, in vitro, and in vivo findings further supporting a role for SEZ6 in AD molecular mechanisms.

Methods

Family and patient description

The family’s pedigree is reported in Fig. 1. We extracted DNA for exome sequencing analysis from the members indicated by the code PR (seven subjects). We had clinical details about three generations after the founder. Ten dementia cases were reported in the whole pedigree, with an additional member having Parkinson’s disease. The age of onset of neurodegenerative disorders ranged from 60 to 70 years. In the first generation, one early-onset dementia case was reported (age at death, 48 years). In the second generation, 8 of 25 siblings (32%) were diagnosed with AD, with an additional case in the third generation (age at onset 64 years). The remaining siblings of this generation were cognitively normal, aged between 35 and 45 years. Apolipoprotein E genotype (APOE) of available patients was in all cases ε3//ε3 apart from PR5 (ε3//ε4). Two siblings of PR5, diagnosed with AD, had dementia too, but they were unavailable for sampling.
Fig. 1

Pedigree of the Italian family with Alzheimer’s disease. We report clinical information for the last three generations after the founders. Sex, age at sampling, and apolipoprotein E (APOE) genotype of each available family member indicated in the box. The numbers next to subjects with dementia are the age at death. The roman numbers refer to the generation, with the progressive numbers linking to every generation sibling

Sporadic AD cases (n = 9) and cognitively normal elderly control subjects (n = 191) were included for independent evaluation of the SEZ6(R615H) variant frequency by digital droplet PCR (ddPCR).

Patients and healthy control subjects were recruited by the same clinical center, and AD was diagnosed according to international criteria. Healthy control subjects were spouses of patients coming to clinical attention, and they had no sign of neurodegenerative disorders and Mini Mental State Examination (MMSE) scores in the normal range [9].

Exome sequencing and APOE genotyping

The full-exome sequencing of 4811 disease-associated genes (clinical exome) was done starting from 50 ng of DNA diluted in Tris-HCl 10 mM, pH 8.5 (TruSight One Sequencing Panel; Illumina, San Diego, CA, USA), following the manufacturer’s instructions. Briefly, capture-based libraries were prepared by pooling three samples per time. The libraries’ concentrations were calculated using a Qubit® dsDNA High-Sensitivity Assay Kit (Invitrogen, Carlsbad, CA, USA), and the distribution of DNA fragments for each library was evaluated using a high-sensitivity DNA kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Each library was run on a MiSeq platform (Illumina) using a 2 × 150-bp (300 cycles) configuration on a V3 sequencing flow cell.

Data analysis was performed according to best practice from the bioinformatics community. Raw sequence fragments (reads) were aligned to the reference genome (human, build hg19) with the Burrows-Wheeler alignment tool [10], followed by post-processing to recalibrate base call quality scores. Variants were called with the Genome Analysis Toolkit [11, 12, 13], using the HaplotypeCaller method, then annotated with the Variant Effect Predictor [14] and loaded into a specialized database [15] for further analysis. In silico mutation impact predictions were extracted from the dbNSFP database [16]. For computation, we used the “bcbio” pipeline (https://github.com/chapmanb/bcbio-nextgen) running on a high-performance computing platform as part of the Cloud4CaRE project. Data files were uploaded to the European Nucleotide Archive with accession number pending.

Selection of candidate variants used the following criteria: (a) depth at least 30×; (b) low frequency in the general population (< 1% in the 1000 Genomes Project); (c) at least a damaging predicted effect as reported from the dbNSFP; and (d) present in all family members affected by AD or their offspring. The APOE genotype was assessed by restriction fragment length polymorphism using the CfoI (Roche, Basel, Switzerland) restriction enzyme, as previously described [17].

Exome sequencing validation by digital droplet PCR

ddPCR experiments were done with the Bio-Rad QX200TM ddPCR system (Bio-Rad Laboratories, Hercules, CA, USA). The mutational assay for SEZ6 R615H was carried out according to the manufacturer’s instructions. Briefly, the TaqMan™ reaction mix, composed of 2× ddPCR Supermix for probes (no deoxyuridine triphosphate), 20× custom target probes for mut SEZ6 (probe sequence: CTACGGTCATGGGCAG-FAM), and 20× reference probes for wild-type SEZ6 (probe sequence: CTACGGTCGTGGGCA-HEX), was assembled at a final concentration of 450 nM and 20 ng of DNA in a volume of 20 μl. This reaction mix was added to a DG8 cartridge together with 60 μl of droplet generation oil for probe and used for droplet generation (QX200 droplet generator; Bio-Rad Laboratories). Droplets were then manually transferred to 96-well PCR plates and placed on a thermal cycler (T100 Thermal Cycler; Bio-Rad Laboratories) for the PCR amplification (thermal cycling conditions: 95 °C for 5 min, 95 °C for 30 s, and 55 °C for 1 min, 40 cycles; 98 °C for 10 min and 4 °C infinite; ramping rate 2 °C/s). The PCR plate was then transferred into the QX100 Droplet Reader for the fluorescence measurement of FAM and HEX probes. The numbers of positive and negative droplets were used to calculate the concentrations (copies/μl) of the target and the reference SEZ6 DNA sequence and their Poisson-based 95% CIs, excluding reactions with fewer than 10,000 total events (positive and negative) (QuantaSoft Analysis pro software 1.0.596; Bio-Rad Laboratories).

For family members and patients with sporadic AD, experiments were run in duplicate; the assay on the healthy population was run once.

Cloning and overexpression of SEZ6(R615H) in H4-SW cells

pSEZ6(R615H) cloning

Synthetic SEZ6(R615H) complementary DNA was provided by GenScript® in pCDNA3.1(+) vector and expanded in competent Escherichia coli cells (strain JM109; Promega, Madison, WI, USA). After purification, pSEZ6(R615H) was verified through the unique enzymatic restriction site PmeI (New England Biolabs, Hitchin, UK) and agarose gel electrophoresis.

Cell culture

H4-SW neuroglioma cells overexpressing human APP gene harboring the Swedish (SW) mutation [18] were grown in DMEM supplemented with 10% FBS, 2 mM l-glutamine, and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, 300 μg/ml hygromycin B, 10 μg/ml blasticidin-S).

Transient transfection was done using FuGENE® HD Transfection Reagent (Promega), and cells were selected with G418 (1200 μg/ml) after 48 h. For clonal selection of SEZ6(R615H) mutants, we picked colonies and analyzed DNA and protein extracts by PCR and Western blotting. Finally, a single-point mutation (G→A) leading to R615H substitution was checked by Sanger sequencing.

PCR for SEZ6(R615H) expression in H4-SW cells

PCR was run in a 20-μl mixture containing 50 ng of DNA, 0.5 mM each of forward primer 5′-CTACGGTCATGGGCAGGATTG-3′, which contains the single-point mutation (G→A), and the reverse oligonucleotide primer 5′- ATCATGGCAGGTGAGGATGGACT-3′ (metabion, Planegg, Germany); 1× PCR buffer 200 mM Tris-HCl, 500 mM KCl (Thermo Fisher Scientific, Waltham, MA, USA); 2.5 mM deoxynucleotide triphosphate (Thermo Fisher Scientific); 25 mM MgCl2 (Thermo Fisher Scientific); and 1 unit of Taq polymerase (Thermo Fisher Scientific). Amplification was done with an initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 61.7 °C for 30 s, extension at 72 °C for 70 s, and a final 5-min extension at 72 °C. The resulting PCR fragments were resolved by 1% agarose gel electrophoresis (Sigma-Aldrich, St. Louis, MO, USA).

Western blotting for SEZ6 overexpression in H4-SW cells

To assess protein overexpression of SEZ6 in H4-SW, protein extracts (18 μg) were separated on 8% SDS-PAGE gel and transferred to a nitrocellulose membrane. Blots were developed using horseradish peroxidase-conjugated secondary antibodies and the ECL chemiluminescence system (MerckMillipore, Burlington, MA, USA). All blots were normalized to α-tubulin and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The following antibodies were used: anti-α-tubulin (1:7500; Abcam, Cambridge, UK) and anti-SEZ6 (1:1000; Aviva Systems Biology, San Diego, CA, USA).

DNA sequencing

To verify the presence of the single point mutation, we amplified the region containing the mutated base by PCR with forward primer 5′- GAGATCACAGACTCGGCTG-3′ and the reverse primer 5′- ATCATGGCAGGTGAGGATGGACT-3′ (metabion). The total amount of the generated PCR product was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and sent to a Sanger sequencing service (Eurofins Genomics, Ebersberg, Germany). Output data were analyzed using Chromas Lite 2.01 software.

Aβ(1–42) and Aβ(1–40) in H4-SW cells expressing SEZ6(R615H)

A specific sandwich enzyme-linked immunosorbent assay (ELISA) (Immuno-Biological Laboratories Co., Gunma, Japan) was used to measure Aβ(1–42) and Aβ(1–40) concentrations in conditioned media from cultured H4-SW cells. A total of 150 × 103 cells were seeded in a six-well plate and grown overnight. The next day, the medium was changed, and after 48 h it was collected and immediately frozen after the addition of a broad-spectrum protease inhibitor (Sigma-Aldrich). An aliquot of 100 μl was used for ELISA to assess each value in triplicate.

Western blot analysis for Sez6 brain expression in 3xTG-AD mice

For Sez6 brain expression analysis, we used 3xTG-AD mice at 3, 9, and 19 months of age. This triple-transgenic model harbors human PS1(M146 V), APP(SW), and MAPT(P301L) transgenes, and starting from around 9 months of age, mice develop at brain level amyloid plaques and protein tau tangles. They also show early signs of synaptic dysfunction (starting from around 3 months of age), including long-term potentiation alteration [19]. Strain, age, and sex-matched nontransgenic animals were used as controls. Mice were housed at 23 °C room temperature with food and water ad libitum and a 12-h/12-h light/dark cycle. To obtain brain protein extract, the cortex was dissected from a single brain hemisphere and homogenized with ice-cold lysis buffer (pH 7.4) containing 1% Triton X-100 and a broad-range protease inhibitor cocktail. Cortex protein extract (20 μg) was analyzed as described above.

Statistics

Data analysis was done using Prism® version 6.0 software (GraphPad Software, La Jolla, CA, USA). In vitro and in vivo data were compared using one-way analysis of variance followed by Tukey’s post hoc test. Two-tailed levels of significance were used, and p < 0.05 was considered significant.

Results

Exome sequencing and APOE genotyping

To identify variants linked to dementia phenotype, we sequenced DNA samples from family members (healthy and AD cases) and unrelated patients with sporadic AD for a set of over 4000 genes reported as implicated in rare and genetic diseases. Our initial analysis identified 15,745 variants passing our quality control filters (variant depth 30× or more). Many of these were common polymorphisms present in the general population, so we selected only those rare in the European population (< 1% frequency), lowering the count to 612 (Additional file 1: Table S1).

To further narrow the search for variants of interest, we used in silico analysis to restrict our findings to those predicted as damaging for protein, finding 138 variants (Additional file 1: Table S1). The majority (96.4%) of possible damaging variants were common between both familial and sporadic AD samples. On the contrary, five variants (3.6%) were exclusive to the family samples (Table 1). In particular, a missense variant in the SEZ6 neuronal gene (c.1844G>A, R615H) was present only in the two available AD cases (PR1 and PR5) and in a first-degree relative (PR2, son of PR5). This variant was localized on one of the extracellular CUB domains of the protein [20, 21] and was predicted to have a high damaging potential (Combined Annotation Dependent Domain [CADD] score = 23). This prompted us to further focus on this variant.
Table 1

Variants exclusive of family members and satisfying the filtering criteria

Chr

Position

Gene

Variant

Amino acid change

(%)

dbSNP

ID

Found in (family code)

chr8

144,589,984

ZC3H3

c.1646

C > T

p.Ser549

Leu

0.5%

rs

149,025,999

PR 1, PR 2, PR 3, PR 4, PR 5, PR 7, PR 9

chr9

738,341

KANK1

c.3391

G > C

p.Ala1131Pro

0.1%

rs

180,816,986

PR1, PR3, PR5

chr17

27,286,417

SEZ6

c.1844

G > A

p.Arg615

His

0.01%

rs

371,753,097

PR1, PR2, PR5

chr20

57,598,807

TUBB1

c.326

G > A

p.Gly109

Glu

0.2%

rs

41,303,899

PR1, PR2, PR3, PR5

chr22

24,717,509

SPECC1L

c.562

C > T

p.Leu188

Phe

0.9%

rs

56,168,869

PR1, PR2, PR3, PR5, PR9

Chr Chromosome number

Percentage population frequency refers to data of the European population frequency derived from the 1000 Genomes Project at the time the manuscript was written. See the “Methods” section of text for further details. Chromosome positions refer to the hg19 assembly. The gene of interest (SEZ6) is highlighted in bold, and members affected with AD are Italic

Validation of exome sequencing SEZ6(R615H) data and variant screening in sporadic AD cases and healthy control subjects

Because the clinical exome results indicated a mutation in SEZ6 gene (c.1844G>A) as unique to the available family members with AD, we performed an independent validation to confirm the result. Using ddPCR, we tested for the SEZ6 variant in exome sequencing-positive family members (n = 3) and in sporadic AD cases (n = 9). To exclude the possibility that the polymorphic variant of SEZ6 identified could be detected at low frequency in the healthy population, too, the mutational assay was also done in a control group of 191 cognitively healthy people.

Figure 2 shows SEZ6 mutational analysis of three family members (PR1, PR2, and PR5) and a representative case of sporadic AD (PR11). Wild-type SEZ6 (green droplets) was detected in all samples, whereas mutated SEZ6 (blue) was detected only in the PR1, PR2 and PR5 samples. A single event with both wild-type and mutated SEZ6 was detected in PR11, probably a polymerase artifact.
Fig. 2

Digital droplet PCR validation of the exome sequencing data. For each patient, a 2D dot plot is shown, reporting the distribution of fluorescence (on the y-axis FAM amplitude, and on the x-axis HEX amplitude). FAM and HEX are the fluorescent dyes for the SEZ6 mutant and SEZ6 wild type, respectively. On the basis of the fluorescence measurements and the droplet distributions, thresholds (pink lines) were set to 5000 for the FAM channel (y-axis) and 3000 for the HEX channel (x-axis). Negative droplets (gray), FAM-positive (blue), HEX-positive (green), and FAM/HEX double-positive (orange) droplets are reported for the four cases and no-template control (NTC) analyzed. Each case represents the sum of independent reactions

Regarding a quantitative measure of the SEZ6 variant, Table 2 reports the concentration as the number of target molecules/μl of wild-type and mutant SEZ6 in all sporadic cases (n = 9), in family members (n = 3), and in healthy individuals (n = 191). Wild-type SEZ6 copies were detected in all groups. The means of wild-type SEZ6 copies/μl were 564, 258, and 130 in the healthy control group, sporadic AD cases, and family members, respectively. A high concentration of mutant SEZ6 was detected in family member samples. The simultaneous presence of the wild-type and the mutated form of SEZ6, with ratios (mutated SEZ6 to wild-type SEZ6) ranging from 0.95 to 1.1, confirmed the heterozygous nature of the SEZ6 C>T 27,286,417–27,186,418 substitution.
Table 2

Mutant SEZ6 assay by digital droplet PCR in healthy control subjects, patients with sporadic Alzheimer’s disease, and family members

Healthy population (n = 191)

Sporadic AD cases (n = 9)

Family members (n = 3)

Sample

Target

Concentration

(copies/μl)

Target

Concentration

(copies/μl)

Sample

Target

Concentration (copies/μl)

Target

Concentration (copies/μl)

Sample

Target

Concentration

(copies/μl)

Target

Concentration (copies/μl)

RATIO (mut/wt)

6

MUT SEZ6

N.D.

WT SEZ6

17

PR3

MUT SEZ6

N.D.

WT SEZ6

239

PR1

MUT SEZ6

116

WT SEZ6

103

1.13

7

MUT SEZ6

N.D.

WT SEZ6

14

MUT SEZ6

N.D.

WT SEZ6

226

MUT SEZ6

114

WT SEZ6

120

0.95

8

MUT SEZ6

N.D.

WT SEZ6

11

PR4

MUT SEZ6

N.D.

WT SEZ6

220

PR2

MUT SEZ6

130

WT SEZ6

130

1.00

9

MUT SEZ6

N.D.

WT SEZ6

59

MUT SEZ6

N.D.

WT SEZ6

224

MUT SEZ6

131

WT SEZ6

127

1.03

11

MUT SEZ6

N.D.

WT SEZ6

41

PR6

MUT SEZ6

N.D.

WT SEZ6

267

PR5

MUT SEZ6

149

WT SEZ6

153

0.97

12

MUT SEZ6

N.D.

WT SEZ6

49

MUT SEZ6

N.D.

WT SEZ6

261

MUT SEZ6

154

WT SEZ6

152

1.01

13

MUT SEZ6

N.D.

WT SEZ6

36

PR7

MUT SEZ6

N.D.

WT SEZ6

331

      

14

MUT SEZ6

N.D.

WT SEZ6

29

MUT SEZ6

N.D.

WT SEZ6

371

      

16

MUT SEZ6

N.D.

WT SEZ6

29

PR8

MUT SEZ6

N.D.

WT SEZ6

307

      

17

MUT SEZ6

N.D.

WT SEZ6

27

MUT SEZ6

N.D.

WT SEZ6

303

      

18

MUT SEZ6

N.D.

WT SEZ6

43

PR9

MUT SEZ6

N.D.

WT SEZ6

254

      

19

MUT SEZ6

N.D.

WT SEZ6

30

MUT SEZ6

N.D.

WT SEZ6

266

      

21

MUT SEZ6

N.D.

WT SEZ6

35

PR10

MUT SEZ6

N.D.

WT SEZ6

239

      

22

MUT SEZ6

N.D.

WT SEZ6

53

MUT SEZ6

N.D.

WT SEZ6

273

      

23

MUT SEZ6

N.D.

WT SEZ6

37

PR11

MUT SEZ6

N.D.

WT SEZ6

233

      

24

MUT SEZ6

N.D.

WT SEZ6

45

MUT SEZ6

N.D.

WT SEZ6

228

      

25

MUT SEZ6

N.D.

WT SEZ6

32

PR12

MUT SEZ6

N.D.

WT SEZ6

212

      

27

MUT SEZ6

N.D.

WT SEZ6

26

MUT SEZ6

N.D.

WT SEZ6

190

      

28

MUT SEZ6

N.D.

WT SEZ6

47

           

29

MUT SEZ6

N.D.

WT SEZ6

48

           

30

MUT SEZ6

N.D.

WT SEZ6

30

           

34

MUT SEZ6

N.D.

WT SEZ6

30

           

36

MUT SEZ6

N.D.

WT SEZ6

49

           

38

MUT SEZ6

N.D.

WT SEZ6

32

           

39

MUT SEZ6

N.D.

WT SEZ6

34

           

41

MUT SEZ6

N.D.

WT SEZ6

74

           

42

MUT SEZ6

N.D.

WT SEZ6

43

           

44

MUT SEZ6

N.D.

WT SEZ6

53

           

46

MUT SEZ6

N.D.

WT SEZ6

64

           

51

MUT SEZ6

N.D.

WT SEZ6

55

           

52

MUT SEZ6

N.D.

WT SEZ6

19

           

53

MUT SEZ6

N.D.

WT SEZ6

32

           

60

MUT SEZ6

N.D.

WT SEZ6

46

           

61

MUT SEZ6

N.D.

WT SEZ6

44

           

62

MUT SEZ6

N.D.

WT SEZ6

64

           

64

MUT SEZ6

N.D.

WT SEZ6

55

           

66

MUT SEZ6

N.D.

WT SEZ6

45

           

67

MUT SEZ6

N.D.

WT SEZ6

46

           

69

MUT SEZ6

N.D.

WT SEZ6

48

           

70

MUT SEZ6

N.D.

WT SEZ6

45

           

71

MUT SEZ6

N.D.

WT SEZ6

67

           

72

MUT SEZ6

N.D.

WT SEZ6

57

           

74

MUT SEZ6

N.D.

WT SEZ6

54

           

89

MUT SEZ6

N.D.

WT SEZ6

47

           

90

MUT SEZ6

N.D.

WT SEZ6

78

           

91

MUT SEZ6

N.D.

WT SEZ6

64.3

           

101

MUT SEZ6

N.D.

WT SEZ6

283

           

112

MUT SEZ6

N.D.

WT SEZ6

524

           

113

MUT SEZ6

N.D.

WT SEZ6

1451

           

114

MUT SEZ6

N.D.

WT SEZ6

962

           

115

MUT SEZ6

N.D.

WT SEZ6

534

           

118

MUT SEZ6

N.D.

WT SEZ6

527

           

119

MUT SEZ6

N.D.

WT SEZ6

1691

           

120

MUT SEZ6

N.D.

WT SEZ6

359

           

129

MUT SEZ6

N.D.

WT SEZ6

186

           

130

MUT SEZ6

N.D.

WT SEZ6

258

           

133

MUT SEZ6

N.D.

WT SEZ6

232

           

135

MUT SEZ6

N.D.

WT SEZ6

373

           

137

MUT SEZ6

N.D.

WT SEZ6

319

           

144

MUT SEZ6

N.D.

WT SEZ6

310

           

151

MUT SEZ6

N.D.

WT SEZ6

396

           

152

MUT SEZ6

N.D.

WT SEZ6

180

           

160

MUT SEZ6

N.D.

WT SEZ6

574

           

162

MUT SEZ6

N.D.

WT SEZ6

400

           

163

MUT SEZ6

N.D.

WT SEZ6

142

           

164

MUT SEZ6

N.D.

WT SEZ6

39

           

170

MUT SEZ6

N.D.

WT SEZ6

96

           

179

MUT SEZ6

N.D.

WT SEZ6

94

           

180

MUT SEZ6

N.D.

WT SEZ6

27

           

182

MUT SEZ6

N.D.

WT SEZ6

1406

           

184

MUT SEZ6

N.D.

WT SEZ6

1994

           

185

MUT SEZ6

N.D.

WT SEZ6

161

           

192

MUT SEZ6

N.D.

WT SEZ6

14.5

           

193

MUT SEZ6

N.D.

WT SEZ6

1740

           

197

MUT SEZ6

N.D.

WT SEZ6

185

           

198

MUT SEZ6

N.D.

WT SEZ6

250

           

199

MUT SEZ6

N.D.

WT SEZ6

145

           

200

MUT SEZ6

N.D.

WT SEZ6

132

           

202

MUT SEZ6

N.D.

WT SEZ6

663

           

205

MUT SEZ6

N.D.

WT SEZ6

658

           

206

MUT SEZ6

N.D.

WT SEZ6

118

           

210

MUT SEZ6

N.D.

WT SEZ6

103

           

212

MUT SEZ6

N.D.

WT SEZ6

23

           

214

MUT SEZ6

N.D.

WT SEZ6

385

           

215

MUT SEZ6

N.D.

WT SEZ6

125

           

219

MUT SEZ6

N.D.

WT SEZ6

223

           

223

MUT SEZ6

N.D.

WT SEZ6

316

           

228

MUT SEZ6

N.D.

WT SEZ6

109

           

233

MUT SEZ6

N.D.

WT SEZ6

385

           

237

MUT SEZ6

N.D.

WT SEZ6

767

           

240

MUT SEZ6

N.D.

WT SEZ6

318

           

241

MUT SEZ6

N.D.

WT SEZ6

15

           

243

MUT SEZ6

N.D.

WT SEZ6

30

           

245

MUT SEZ6

N.D.

WT SEZ6

166

           

247

MUT SEZ6

N.D.

WT SEZ6

161

           

251

MUT SEZ6

N.D.

WT SEZ6

164

           

253

MUT SEZ6

N.D.

WT SEZ6

491

           

254

MUT SEZ6

N.D.

WT SEZ6

772

           

255

MUT SEZ6

N.D.

WT SEZ6

771

           

257

MUT SEZ6

N.D.

WT SEZ6

148

           

261

MUT SEZ6

N.D.

WT SEZ6

875

           

263

MUT SEZ6

N.D.

WT SEZ6

381

           

267

MUT SEZ6

N.D.

WT SEZ6

442

           

270

MUT SEZ6

N.D.

WT SEZ6

368

           

275

MUT SEZ6

N.D.

WT SEZ6

317

           

276

MUT SEZ6

N.D.

WT SEZ6

368

           

277

MUT SEZ6

N.D.

WT SEZ6

186

           

278

MUT SEZ6

N.D.

WT SEZ6

63

           

279

MUT SEZ6

N.D.

WT SEZ6

234

           

287

MUT SEZ6

N.D.

WT SEZ6

99

           

293

MUT SEZ6

N.D.

WT SEZ6

125

           

324

MUT SEZ6

N.D.

WT SEZ6

605

           

325

MUT SEZ6

N.D.

WT SEZ6

153

           

326

MUT SEZ6

N.D.

WT SEZ6

692

           

327

MUT SEZ6

N.D.

WT SEZ6

713

           

328

MUT SEZ6

N.D.

WT SEZ6

391

           

332

MUT SEZ6

N.D.

WT SEZ6

759

           

333

MUT SEZ6

N.D.

WT SEZ6

661

           

337

MUT SEZ6

N.D.

WT SEZ6

798

           

338

MUT SEZ6

N.D.

WT SEZ6

903

           

340

MUT SEZ6

N.D.

WT SEZ6

40

           

341

MUT SEZ6

N.D.

WT SEZ6

274

           

342

MUT SEZ6

N.D.

WT SEZ6

240

           

344

MUT SEZ6

N.D.

WT SEZ6

209

           

345

MUT SEZ6

N.D.

WT SEZ6

873

           

348

MUT SEZ6

N.D.

WT SEZ6

2330

           

350

MUT SEZ6

N.D.

WT SEZ6

387

           

351

MUT SEZ6

N.D.

WT SEZ6

430

           

353

MUT SEZ6

N.D.

WT SEZ6

360

           

360

MUT SEZ6

N.D.

WT SEZ6

473

           

361

MUT SEZ6

N.D.

WT SEZ6

553

           

362

MUT SEZ6

N.D.

WT SEZ6

2470

           

363

MUT SEZ6

N.D.

WT SEZ6

889

           

366

MUT SEZ6

N.D.

WT SEZ6

1990

           

367

MUT SEZ6

N.D.

WT SEZ6

452

           

368

MUT SEZ6

N.D.

WT SEZ6

1736

           

369

MUT SEZ6

N.D.

WT SEZ6

1436

           

375

MUT SEZ6

N.D.

WT SEZ6

588

           

376

MUT SEZ6

N.D.

WT SEZ6

544

           

377

MUT SEZ6

N.D.

WT SEZ6

623

           

401

MUT SEZ6

N.D.

WT SEZ6

803

           

404

MUT SEZ6

N.D.

WT SEZ6

494

           

406

MUT SEZ6

N.D.

WT SEZ6

200

           

407

MUT SEZ6

N.D.

WT SEZ6

482

           

408

MUT SEZ6

N.D.

WT SEZ6

105

           

409

MUT SEZ6

N.D.

WT SEZ6

3260

           

418

MUT SEZ6

N.D.

WT SEZ6

190

           

422

MUT SEZ6

N.D.

WT SEZ6

1325

           

430

MUT SEZ6

N.D.

WT SEZ6

772

           

434

MUT SEZ6

N.D.

WT SEZ6

1233

           

435

MUT SEZ6

N.D.

WT SEZ6

1844

           

440

MUT SEZ6

N.D.

WT SEZ6

90

           

446

MUT SEZ6

N.D.

WT SEZ6

745

           

451

MUT SEZ6

N.D.

WT SEZ6

1366

           

453

MUT SEZ6

N.D.

WT SEZ6

1185

           

454

MUT SEZ6

N.D.

WT SEZ6

2950

           

466

MUT SEZ6

N.D.

WT SEZ6

329

           

468

MUT SEZ6

N.D.

WT SEZ6

681

           

493

MUT SEZ6

N.D.

WT SEZ6

80

           

497

MUT SEZ6

N.D.

WT SEZ6

154

           

499

MUT SEZ6

N.D.

WT SEZ6

128

           

501

MUT SEZ6

N.D.

WT SEZ6

1814

           

511

MUT SEZ6

N.D.

WT SEZ6

547

           

512

MUT SEZ6

N.D.

WT SEZ6

48.2

           

513

MUT SEZ6

N.D.

WT SEZ6

40.8

           

514

MUT SEZ6

N.D.

WT SEZ6

1019

           

519

MUT SEZ6

N.D.

WT SEZ6

1382

           

520

MUT SEZ6

N.D.

WT SEZ6

791

           

521

MUT SEZ6

N.D.

WT SEZ6

1858

           

522

MUT SEZ6

N.D.

WT SEZ6

2180

           

523

MUT SEZ6

N.D.

WT SEZ6

1849

           

531

MUT SEZ6

N.D.

WT SEZ6

2110

           

532

MUT SEZ6

N.D.

WT SEZ6

3030

           

535

MUT SEZ6

N.D.

WT SEZ6

1096

           

537

MUT SEZ6

N.D.

WT SEZ6

1941

           

538

MUT SEZ6

N.D.

WT SEZ6

78

           

539

MUT SEZ6

N.D.

WT SEZ6

917

           

542

MUT SEZ6

N.D.

WT SEZ6

1650

           

543

MUT SEZ6

N.D.

WT SEZ6

937

           

545

MUT SEZ6

N.D.

WT SEZ6

1423

           

546

MUT SEZ6

N.D.

WT SEZ6

818

           

549

MUT SEZ6

N.D.

WT SEZ6

1196

           

550

MUT SEZ6

N.D.

WT SEZ6

716

           

558

MUT SEZ6

N.D.

WT SEZ6

845

           

567

MUT SEZ6

N.D.

WT SEZ6

724

           

570

MUT SEZ6

N.D.

WT SEZ6

765

           

571

MUT SEZ6

N.D.

WT SEZ6

2290

           

574

MUT SEZ6

N.D.

WT SEZ6

790

           

575

MUT SEZ6

N.D.

WT SEZ6

1399

           

578

MUT SEZ6

N.D.

WT SEZ6

1293

           

580

MUT SEZ6

N.D.

WT SEZ6

947

           

ND Not detectable

Alzheimer’s disease cases are underlined. For each group, patient code, digital droplet PCR target, and the calculated concentration (copies/μl) are reported. For the last group, the ratio, defined as concentration mutant SEZ6/concentration wild-type SEZ6, is also reported

Aβ peptide generation in H4-SW cells

Three different H4-SW stable clonal lines (C3, C4, and C13) transfected with a pCDNA3.1 plasmid coding for SEZ6(R615H) mutant were selected, and the presence of the variant at DNA level was confirmed by allele-specific PCR and sequencing (data not shown). The effect of the R615H substitution on Aβ(1–42) and Aβ(1–40) production by H4-SW cells was assessed in conditioned media from cultured H4-SW(R615H) in comparison to H4-SW cells (untransfected or mock-transfected with an empty pCDNA3.1 vector) (Fig. 3a). The mean concentration of released Aβ(1–42), normalized to cell total protein content, was significantly lower in C4 and C13 than in controls, whereas for the C3 line, there was a trend in the same direction (p = 0.07). The Aβ(1–40) assay showed no differences (Fig. 3b).
Fig. 3

Evaluation of SEZ6 relevance for Alzheimer’s disease (AD) mechanisms in in vitro and in vivo models. a Quantification by enzyme-linked immunosorbent assay of soluble amyloid-β 1–40 (Aβ1–40) in conditioned media from H4-SW clonal lines (C3, C4, and C13) overexpressing SEZ6(R615H). The amyloid peptide concentration was normalized to the total protein content of the producing cells of each replicate. Measures are the mean ± SD of three independent wells. H4-SW Untransfected control; Ø H4-SW control transfected with pCDNA3.1 empty vector. b Same as in (a) except for the assessment of Aβ1–42 soluble form. * p < 0.05; *** p < 0.001, one-way analysis of variance (ANOVA) and post hoc test; # p < 0.05 vs. C4 and p < 0.01 vs. C13, one-way ANOVA and post hoc test. c Representative Western blotting for Sez6 protein detection in brain cortical extract from 3xTG-AD mice. Mice were killed at ages 3, 9, or 19 months, and Sez6 expression was assessed in transgenic and matched nontransgenic (NTG) animals. Each group was composed of three mice, and every animal was loaded in duplicate in the SDS-PAGE experiment. * Unspecific signal. d Densitometric quantification of all Western blot analysis data for Sez6 protein cortical expression (n = 3 mice/group) using ImageJ software. Each signal was normalized to the corresponding α-tubulin band to control for unequal protein loading. Results are expressed as a percentage of the youngest group (3 months) * p < 0.05, one-way ANOVA and post hoc test. mo. Months from birth

Sez6 brain expression in 3xTG-AD mice

Given that few experimental data linked SEZ6 to AD, we also examined murine Sez6 expression in a transgenic line model of AD (3xTG-AD), in comparison with age-matched nontransgenic controls (NTG) (Fig. 3c and d). Mice were killed at ages ranging from 3 to 19 months, and Sez6 protein expression was assessed at brain cortical level. Sez6 protein markedly decreased with age, particularly between 3 and 19 months. However, this reduction was common to both the 3xTG-AD and NTG lines and thus not unique to the AD model.

Discussion

Pathogenic mutations in APP, PSEN1, or PSEN2 genes are linked to FAD [3, 4]. PSEN1 mutations are responsible for about 60% of the genetic cases of AD, and 286 pathogenic variants have been described in the three above-cited genes [22]. We report an Italian family with AD that we previously screened by denaturing high-performance liquid chromatography (data not shown) for APP, PSEN1, or PSEN2 mutations with no results. Considering that rare variants in other genes have been associated with AD [7], we decided to perform targeted exome-sequencing analysis that yielded a large number of variants; in order to identify those closely related to the disease, we employed a recursive filtering strategy. This strategy was based on the removal of high-frequency (> 1%) variants using a public database (1000 Genomes Project) with in silico prediction software (SIFT, PolyPhen2, CADD) to exclude potentially harmless mutations and focus on variants present in FAD but not sporadic AD samples. We gave priority to the SEZ6(R615H) variant among those reported in Table 1, considering that SEZ6 has already been reported as relevant for molecular mechanisms involved in AD pathogenesis, because it is a substrate of the BACE-1 enzyme (β-secretase), affects synapse formation, and is reduced in the cerebrospinal fluid of patients with AD, as revealed by a proteomic study [23, 24, 25]. SEZ6 gene mutations have been also reported in association with febrile seizures, and SEZ6 was proposed as a candidate gene for epilepsy [26, 27]. Moreover, SEZ6 mutations were found in cases of childhood-onset schizophrenia [28]. The rare variant R615H (rs371753097, C/T) was reported in the 1000 Genomes Project as absent in Toscani in Italy (TSI) population and had a frequency in the whole project of 0.0002 [29]. Another interesting genetic variant we found by exome sequencing that is deserving of attention is A1131P in the KANK1 gene [30], which was present in the two AD cases (PR1 and PR5) and in PR3, sibling of PR1. However, PR3 did not have dementia at sampling (age 67 years), and her clinical state is currently unchanged, even though we are not able to exclude a possible later onset. The human KANK1 gene (alias ANKRD15) was originally described to be a tumor suppressor in renal cell carcinoma, and it encodes an ankyrin repeat domain-containing protein (Kank). It belongs to a family of four homologous members that have a role in actin stress fiber formation and renal pathophysiology [31, 32]. There is no reported interaction of KANK1 with SEZ6 or AD-related genes. However, a role of KANK1 mutation or deletion was reported in cerebral palsy spastic quadriplegic type 2, a central nervous system developmental disorder [33]. Moreover, to the best of our knowledge, no data associate KANK1 with AD.

In our study’s family, we were able to correlate the AD pathology to R615H presence, which was found in the two available AD-affected members and one first-degree relative of an AD case, whose age at sampling in 2003 (PR2, 37 years) was far below the family age of onset (range, 60–70 years) to expect clinical signs. The current clinical diagnosis of PR2 (51 years) is unchanged. We also confirmed that R615H frequency is very low (< 1%) in the Italian population, because we were unable to detect the variant in 200 family-unrelated subjects.

Because it is a common finding that AD pathogenic mutations increase Aβ(1–42) peptide generation [34], we examined the effect of the R615H variant in a cell model in this respect. In the H4-SW line, we noticed a decrease in Aβ(1–42), whereas Aβ(1–40) was unchanged. However, the increase of Aβ(1–42) in association with FAD-linked mutations is not always replicated. In fact, some presenilin mutants with proved pathologic action did not increase Aβ(1–42) but acted on other Aβ peptide generation or even had no impact on this proteolytic cleavage. In the latter case, the hypothesis is that the mutation affects important functions of presenilin other than the γ-secretase activity [35, 36]. It is worth underlining that we found a peculiar biochemical effect of the PSEN1 mutation E318G that increased Aβ(1–40) only in cultured skin primary fibroblasts [17]. Our failure to detect an increase of Aβ(1–42) might depend on the reported role of SEZ6 protein as substrate for BACE-1 [23], so its overexpression may be competitive for APP in the cell model tested. We need further experiments to clarify the role of the R615H variant in this context.

Finally, we followed SEZ6 cortical expression in a mouse model of AD (3xTG-AD). Considering that it changed similarly in 3xTG-AD and control mice, we were unable to link this result to AD-specific patterns, but we did notice a decrease of SEZ6 protein with age, in agreement with this gene’s reported role in brain development [37, 38]. A damaging mutation (as R615H is predicted to be) may have an impact on the protein activity from birth, with possible neuropathologic outcomes, likely in combination with other triggering factors, also considering the reported role of SEZ6 in dendritic spine dynamics and cognition [39].

This study has limitations, mainly linked to the unavailability of genomic DNA from all the family’s AD-affected members alive at sampling. Moreover, we decided to use a targeted exome-sequencing strategy that, on one hand, gave us clinical data supporting a rational choice of candidate variants to be prioritized, but on the other hand, prevented us from ruling out that additional coding mutations in genes not included in our panel may be linked to AD phenotype, thus acting in synergy with SEZ6 (R615H).

Conclusions

In summary, by using a targeted exome-sequencing approach, we discovered a rare SEZ6 variant exclusive to AD members of a large Italian family carrying no typical FAD-linked mutations that might have a role in disease onset, in particular taking into account the already described involvement of SEZ6 in AD pathogenic mechanisms linked to amyloid-β (A4) precursor protein (APP) and brain physiology, even though the exact molecular pathway linking SEZ6 to AD is still unclear.

Notes

Acknowledgements

We are grateful to the family that kindly participated in this study. We thank Judith Baggott for English-language editing.

Funding

This work was supported by Fondazione Italo Monzino (Milan, Italy).

Availability of data and materials

Exome sequencing data files were uploaded to the European Nucleotide Archive (https://www.ebi.ac.uk/ena) with accession numbers pending.

Authors’ contributions

LP performed the digital droplet PCR assay and exome sequencing. LBe alanyzed genomic data and produced bioinformatics output. FF and LBo prepared the H4-SW clonal lines and measured SEZ6 gene expression in transgenic mice. PC recruited the families with sporadic Alzheimer’s disease and control subjects. DA, SM, and GF drafted the manuscript. All authors critically revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All patients or their relatives gave informed consent for participation in this study, which was approved by the local ethics committee at Istituto di Neurologia, Università di Parma (PC), under the responsibility of Prof. Paolo Caffarra. Animal studies were run in compliance with national laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law (D.lgs 26/2014; authorization no. 19/2008-A, issued March 6, 2008, by the Ministry of Health); Mario Negri institutional regulations and policies providing internal authorization for people conducting animal experiments (Quality Management System Certificate UNI EN ISO 9001:2008 registration no. 6121); the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (2011 edition), and European Union directives and guidelines (EEC Council Directive 2010/63/UE). The Statement of Compliance (Assurance) with the Public Health Service (PHS) Policy on Human Care and Use of Laboratory Animals was recently reviewed (September 9, 2014) and will expire on September 30, 2019 (Animal Welfare Assurance no. A5023-01, DL. vo 116/1992, Gazzetta Ufficiale, Suppl. 40, Feb.18, 1992; Circolare nr. 8, Gazzetta Ufficiale, July 14, 1994) and international laws and policies (EEC Council Directive 86/609, OJL 358, 1, Dec.12, 1987; NIH Guide for the Care and Use of Laboratory Animals 8th edition, 2011).

Consent for publication

Not applicable, because this article does not contain any individual person’s data, images, or videos.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

13195_2018_435_MOESM1_ESM.xlsx (86 kb)
Additional file 1: Table reporting the sequencing results of DNA from PR family members and unrelated sporadic AD cases, including only rare variants in the European population (frequency less than 1%) [low frequency page]. The same reults were further filtered to show variants with predicted damaging action [predicted damage page]. (XLSX 85 kb)

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Department of OncologyIstituto di Ricerche Farmacologiche Mario Negri IRCCSMilanItaly
  2. 2.Dipartimento di Chimica, Materiali e Ingegneria Chimica “G. Natta”, Politecnico di MilanoMilanItaly
  3. 3.Department of NeuroscienceIstituto di Ricerche Farmacologiche Mario Negri IRCCSMilanItaly
  4. 4.Department of Neuroscience, Istituto di NeurologiaUniversità di ParmaParmaItaly

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