Biallelic Loss of Function of SORL1 in an Early Onset Alzheimer’s Disease Patient

Abstract

Heterozygous SORL1 protein truncating variants (PTV) are a strong risk factor for early-onset Alzheimer’s disease (EOAD). In case control studies performed at the genome-wide level, PTV definition is usually straightforward. Regarding splice site variants, only those affecting canonical sites are typically included. Some other variants, not annotated as PTV, could, however, affect splicing and hence result in a loss of SORL1 function. We took advantage of the whole exome sequencing data from the 9/484 patients with a previously reported SORL1 PTV in the French EOAD series and searched for a second variant which may affect splicing and eventually result in more than 50% loss of function overall. We found that one patient, known to carry a variant predicted to disrupt the canonical 5’ splice site of exon 8, also carried a second novel intronic variant predicted to affect SORL1 splicing of exon 29. Segregation analysis showed that the second variant was located in trans from the known PTV. We performed ex vivo minigene splicing assays and showed that both variants led to the generation of transcripts containing a premature stop codon. This is therefore the first evidence of a human carrying biallelic SORL1 PTV. This patient had a family history of dementia in both maternal and paternal lineages with later ages of onset than the proband himself. However, his 55 years age at onset was in the same ranges as previously published SORL1 heterozygous PTV carriers. This suggests that biallelic loss of SORL1 function is an extremely rare event that was not associated with a dramatically earlier age at onset than heterozygous SORL1 loss-of-function variant carriers, in this single patient.

Keywords

Compound heterozygous early-onset Alzheimer’s disease haploinsufficiency knockout recessive SORL1

INTRODUCTION

During the past six years, knowledge about Alzheimer’s disease (AD) genetic component has increased significantly thanks to the advent of massive parallel sequencing (for review, see [1]). Besides rare autosomal dominantly-inherited forms caused by PSEN1, PSEN2, or APP mutations, AD is considered as a complex trait with a high genetic component. Rare damaging variants in three genes have recently been shown to be associated with AD [2]: TREM2 [3 –5], SORL1 [6 –8], and ABCA7 [9 –11]. In a case-control analysis using whole-exome sequencing (WES) data, we have previously shown that rare heterozygous protein-truncating (PTV) and missense SORL1 variants predicted to be damaging were a major risk factor for early-onset Alzheimer’s disease (EOAD, onset before 65 years), with an odds ratio (OR) as high as 5.03 (95% confidence interval (CI) [2.02–14.99]); OR was 8.86 [3.35–27.31] after restriction to patients with a positive family history of AD) [6].

SORL1 (Sortilin-related receptor, also known as sortilin-related receptor, L(DLR class) A repeats-containing) has two main functions influencing the secretion of the amyloid-β (Aβ) peptide: 1) it binds to the amyloid-β protein precursor (AβPP) and can redirect it toward a non-amyloidogenic pathway [12] and 2) it binds to nascent Aβ peptides and direct them to the lysosome, precluding their secretion [13]. Nine of the variants identified in our study were classified as PTV, i.e., nonsense, frameshift, and canonical splice site variants, all predicted to introduce a premature stop codon. The existence of nonsense-mediated decay (NMD) was assessed through the analysis of RNA isolated from the blood of 3 French and one Belgian PTV carriers in different studies, showing reduced expressions of the mutated alleles, which is consistent with a mechanism of NMD [6 , 14]. This suggests that haploinsufficiency is a major mechanism leading to increased Aβ secretion in SORL1 PTV carriers.

Most case-control analyses performed at the exome level, including ours, focused on variants with a straightforward PTV annotation. Regarding splice site variants, a proportion of them can introduce a premature stop codon (out of frame splicing) and another proportion keeps the reading frame intact, although removing a given number of codons. Variants disrupting canonical splice sites are, however, generally regarded as PTV as a whole in association studies. In such analyses, only the variants affecting the canonical splice sites (–2, –1, +1, or +2 bp in flanking introns) were included [6]. However, some single nucleotide variations (SNV) or short insertions or deletions (indels) located outside the canonical splice sites can also affect splicing through diverse mechanisms, e.g., the modification or disruption of a canonical splice site, the introduction of a novel splice site, the activation of a cryptic site, or the alteration of splicing regulatory elements (SRE) [15]. Some of the effects can be predicted by in silico tools. However, the accuracy of these bioinformatics programs is still insufficient, especially those dedicated to SRE predictions, and the outcomes on transcripts of such splicing modifications are in some cases difficult to apprehend on the sole basis of in silico data. For these reasons, it is not possible to firmly conclude about any effect without accurate experimental assessment. Hence, this is a real challenge to include rare SNVs/indels located outside the canonical splice sites but predicted to affect splicing in case-control studies performed at the exome level.

All SORL1 PTV have been reported to be carried in a heterozygous state. Given the importance of SORL1 haploinsufficiency in EOAD genetic determinism and the high number of variants of unknown significance among the synonymous, intronic, and missense SORL1 variants, we decided to focus on SORL1 PTV carriers to detect rare variants affecting splicing in the second SORL1 allele and which may overall result in more than 50% loss of function. For that purpose, we took advantage from WES data from the 9/484 previously published patients from our series carrying one SORL1 PTV and identified one patient with bi-allelic SORL1 loss of function.

METHODS

Patients

Of the 484 unrelated EOAD patients of the French series reported before [6], we selected the 9 patients carrying one PTV (patients and variant information are provided in Supplementary Table 1). All patients gave informed written consent for genetic analyses and this study was approved by the CPP Ile de France II ethics committee. Of note, none of them was a carrier either of a pathogenic or likely pathogenic APP, PSEN1, or PSEN2 variant, or of an APP duplication or MAPT duplication [6, 16].

WES data

Generation and analysis of WES data has been described in details previously [6]. We extracted all rare SORL1 variants, i.e., with a minor allele frequency (MAF) <1% in the ExAC database, Non-Finish European (NFE) population [17]. The reference genome used in this study is GRCh37 (hg19) and the reference SORL1 transcript used for variant nomenclature, NM_003105.5. For exon numbering, we used exon 1 as the first coding exon of this transcript.

In silico analysis

The Alamut Visual software (Interactive Biosoftware, Rouen, France; http://www.interactive-biosoftware.com) was used to simulate the effect of variants on splicing using different algorithms. We reported the score of the following three, as in previous work [15]: SpliceSiteFinder-like (SSF, http://www.interactive-biosoftware.com), MAX-EntScan (MES, http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html) and the splice site module of Human Splicing Finder (HSF-ss, http://www.umd.be/HSF/).

For missense variants predictions, we used Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and Mutation Taster (http://www.mutationtaster.org/), as in previous work [6].

Splicing minigene reporter assays

Assays were performed as previously described with minor modification [15]. Wild-type (WT) and mutant genomic fragment of exon 8 and 29 of SORL1 and their close intronic regions (exon 8: c.1042-243_c.1211+228; exon 29: c.3947-339_c.4078+227) were PCR-amplified from patient DNA and inserted into pCAS2 vector by recombination using the Seamless Ligation Cloning extract technology also called SLiCE [18]. Then, WT and mutant minigenes were transfected into Hela cells grown at ∼60% of confluence in 12-well plates using the FuGENE 6 transfection reagent (Roche Applied Science). HeLa cells were cultivated in Dulbecco’s modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum in a 5% CO₂ atmosphere at 37°C. Total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer’s instruction. The minigene transcripts were then reverse transcribed and PCR amplified (30 cycles) from 100 ng of total RNA in a 25μL reaction volume using the OneStep RT-PCR kit (Qiagen) and the appropriate primers (available upon request). RT-PCR products were separated by electrophoresis on 2% agarose gels containing ethidium bromide and visualized by exposure to ultraviolet light under non-saturating conditions using the Gel Doc XR image acquisition system (Bio-RAD). Gel extraction and sequencing of the RT-PCR products were carried out as previously described [15]. Fluorescent RT-PCR reactions were performed using the equivalent primers followed by capillary electrophoresis (Applied Biosystems) and electropherograms were analyzed by using the GeneMapper v5.0 software (Applied Biosystems). RT-PCR products were subcloned using the pGEM-T vector and Sanger sequenced.

For designating aberrant transcripts, we used the symbols Δ for partial or complete exon deletion and ▾ for the inclusion of intronic sequence; p for 3’ splice site shift and q for 5’ splice site shift, as previously described [19].

RESULTS

Analysis of WES data

Among the 9 patients carrying one heterozygous PTV included in our study, one carried 2 additional rare SNVs or indels in SORL1: patient ROU-0055-001, hereafter referred to as patient 001. None of the other 8 SORL1 PTV carriers exhibited a second rare variant.

Patient 001 carried the previously reported c.1211+2T>G canonical splice variant in intron 8. On top of this variant, we identified a c.3947-3insG variant in intron 28 and a c.4265A>G, p.(N1422S) change in exon 31. The c.3947-3insG and the c.4265A>G, p.(N1422S) were not reported before as they were out of the inclusion criteria for the association study because of the position further than -2 in the intron and discordant in silico predictions of missense variants, respectively [6]. Both novel variants were confirmed by Sanger sequencing.

Patient and family description

Patient 001 presented progressive cognitive decline starting from the age of 55 years. His medical history was marked by duodenal ulcer, tobacco use, and arteritis of the lower limbs. Upon neuropsychological examination at the age of 57, Mini-Mental Scale Examination (MMSE) scored 19/30 (N>22 considering education level and age-related normative values) and Mattis Dementia Rating Scale (MDRS), 105/144 (N>119), he showed severe reduction of verbal fluency (semantic fluency 9, N>16; phonemic fluency: 3, N>9), visuoconstructive apraxia (Rey figure copy scored 14/36, N>27.5), and the Weschler memory quotient scored 70. During follow up, cognitive decline was observed: MMSE scored 14 at the age of 60 and 6 at the age of 62, while MDRS scored 91 and then 22 at the same respective ages. Weschler memory quotient was 56 at age 60 and he failed when assessing this scale at age 62. At the age of 63, he exhibited visual delusion, psychomotor agitation, and prosopagnosia. He experienced one epileptic seizure at the age of 64, was bedridden from the age of 66 and deceased at the age of 70 years. The diagnosis of probable AD was made following the NINCDS-ADRDA criteria [20]. His APOE genotype is 3-4. He did not carry any TREM2 or ABCA7 known risk allele or PTV.

His family history was marked by dementia in his both parents. His father deceased in the context of severe dementia at the age of 76 years, starting more than 6 years before (age of onset is not known from the family). The paternal grandfather of patient 001 died at the age of 71 years with severe dementia starting before the age of 65 (Fig. 1). The mother of patient 001 was also diagnosed with dementia with an unknown age of onset. She required attention for daily life skills before the age of 78, age at which she could not live on her own anymore. At the age of 85, she was described as being bedridden with severe dementia and died at the age of 91. No history of dementia was noticed in her own parents, who died at young ages from other causes.

Fig.1

Reduced pedigree of family ROU 0055. The age at death is indicated and the age at dementia onset is in parentheses. Filled symbols represent patients with probable Alzheimer’s disease. The vertical bars represent the SORL1 gene and the genotypes at all three positions where a rare variant has been identified in patient 001, as well as segregation data in his mother and inferred genotype of his father. *APOE N4: inferred partial genotype.

The DNA of the mother, isolated from whole blood, was available for segregation analysis, but the father’s DNA was not available. No necropsy was performed in this family and no RNA sample is available. The analysis of the mother’s DNA showed that she carried both c.3947-3insG and c.4265A>G, p.(N1422S) variants but not the c.1211+2T>G variant, demonstrating that the first two are located in cis and suggesting that the c.1211+2T>G is in trans from the other two in patient 001 (Fig. 1). We confirmed the phasing of these variants through the analysis of DNA isolated from the blood of an asymptomatic relative in the patient’s offspring, carrying the maternal allele without the c.1211+2T>G variant (not shown). The APOE genotype of the proband’s mother is 3-3.

In silico predictions

The c.1211+2T>G variant, located in intron 8, is absent from variant databases including gnomAD [17]. It is predicted to disrupt the canonical 5’ splice site of exon 8. If exon skipping would result from such altered splicing, it would lead to a frameshift (Table 1, Fig. 2). Of note, in silico tools also predict the existence of two cryptic 5’ splice sites that could take over: one exonic located 5 nucleotides (nt) upstream of the natural 5’ splice site and one intronic located 11 nt downstream of the natural 5’ splice site. If used, they would result in the removal of 5 exonic nt or the retention of 11 intronic nt, respectively. All three hypotheses lead to a premature stop codon.

Table 1

Summary of the in silico predictions of the impact on splice sites and on protein of rare SORL1 variants identified in patient 001

				Nearest canonical splice site					Cryptic splice site			Protein level predictions
cDNA nomenclature	Protein level nomenclature	gnomAD allele frequency*	Exon/intron	Distance (bp)	Type	SSF fold change	MES fold change	HSF-ss fold change	SSF	MES	HSF-ss	SIFT	PolyPhen2 HumDiv	Mutation Taster
c.1211+2T>G	p.?	0	Intron 8	+2	5’ ss	–100%	–100%	–100%	No effect on nearest cryptic sites	No effect on nearest cryptic sites	No effect on nearest cryptic sites	NA	NA	NA
c.3947-3insG	p.?	0	Intron 28	–3	3’ ss	–12.3%	–100%	–10.7%	No effect on nearest cryptic sites	No effect on nearest cryptic sites	No effect on nearest cryptic sites	NA	NA	NA
c.4265A>G	p.(N1422S)	1.6 10^–5	Exon 31	+52	3’ ss	0	0	0	New cryptic 3’ss:	New cryptic 3’ss:	New cryptic 3’ss:	Tolerated	Probably Damaging	Disease causing
									Score = 72.4	Score = 5.6	Score = 84.7

cDNA, protein level nomenclatures and exon/intron numbering refer to transcript NM_003105.5. SSF, SpliceSiteFinder-like; MES, MAX-EntScan; HSF-ss, splice site module of Human Splicing Finder. SSF, MES, and HSF-ss fold change correspond to the percentage of score change compared to the WT sequence at the closest canonical splice site. SSF, MES, and HSF-ss scores range from 0 to 100, 16, and 100, respectively. ^*gnomAD allele frequency in the European non-Finish population. See also Fig. 2.

Fig.2

Screenshots of the Alamut Visual Software showing in silico predictions of the impact of SORL1 variants on splice sites of exon 8 (A), exon 29 (B) and exon 31 (C). The variant c.1211+2T>G (A, lower panel, in red) is predicted to disrupt the canonical 5’ splice site (A, upper panel, within red square). This can result in a skipping of exon 8 or/and the use of exonic and intronic cryptic 5’ splice sites, respectively located 5 nucleotides upstream of the natural 5’ splice site and 11 nucleotides downstream of the natural 5’ splice site (within blue squares). The variant c.3947-3insG (B, lower panel, in red) is predicted to disrupt the canonical 3’ splice site (B, upper panel, within red square). This can result in exon skipping which corresponds to the in frame loss of the 132 bp of exon 30, but the aberrant exon 28 - exon 30 junction would be a premature stop codon. On note, two close cryptic 3’ and 5’ splice sites are predicted, respectively located in c.3947-44_3947-43 (within green square) and c.3970_3971 (within blue square). The variant c.4265A>G, p.(N1422S) (C, lower panel, in red) is predicted to introduce a novel 3’ splice site (within green square, lower panel). If used, it would lead to the loss of the first 52 nucleotides of exon 31, leading to a frameshift.

The variant c.3947-3insG is absent from variant databases including gnomAD. It is predicted to disrupt the canonical 3’ splice site of exon 29. If exon skipping results from such altered splicing, it would lead to an in-frame loss of its corresponding 132 bp in RNA. However, at the aberrant exon 28 - exon 30 junction, the novel codon would be TAA, a premature stop codon (Table 1, Fig. 2). Of note, a cryptic intronic 3’ splice site is predicted 43-44 nt upstream of the natural 3’ splice site.

The variant c.4265A>G, p.(N1422S) has a MAF of 0.0016% in the NFE population from gnomAD and maps in 3’ from the other two variants. If the reading frame of the mRNA is not affected by one of these variants, it would result in an Asparagine to Serine change at position 1422 in the protein, which is predicted to be tolerated by SIFT, disease-causing by Mutation Taster, and probably damaging by PolyPhen2 (Table 1). However, splicing prediction software suggest that a novel 3’ splice site could be created by this variant. If used, it would lead to the loss of 52 bp, leading to a frameshift (Table 1, Fig. 2).

SORL1 compound heterozygous variations result in bi-allelic protein truncation through splicing alterations

As RNA was not available from any affected individual from this family, we carried out ex-vivo splicing assays using pCAS minigenes assessing the paternal c.1211+2T>G and the maternal c.3947-3insG variants. Here, it is important to note that the variants c.3947-3insG and c.4265A>G, p.(N1422S) are located in cis and c.3947-3insG is predicted to result in a frameshift upstream of any putative effect on splicing of the c.4265A>G, p.(N1422S) variant.

The pCAS minigene containing WT exon 8 produced only the transcript with the normal exon inclusion at the expected size (404 nt) as determined by fluorescent RT-PCR and sequencing analyses. In contrast, the same construct with the c.1211+2T>G variation led to the production of three major products with different size (234, 399 and 415 nt) as ascertained by the fluorescent RT-PCR experiment. None of them corresponded to the natural transcript (Fig. 3, Supplementary Figure 1). Direct sequencing of the RT-PCR products did not allow the identification of the aberrant transcripts because of the presence of heteroduplexes. We therefore cloned and sequenced these three aberrant transcripts and could conclude that they correspond to: 1) the total skipping of exon 8 (transcript Δ8), 2) the deletion of the last 5 nt of exon 8 (transcript Δ8q), consequence of the use of the cryptic exonic 5’ splice site, and 3) the insertion of the first 11 nt of intron 8 (transcript▾8q), consequence of the use of the cryptic intronic splice site (Fig. 3). Transposing these minigene data into the physiological context of SORL1 transcript, these three aberrant transcripts would lead to the introduction of a premature stop codon: p.Glu348Valfs*5, p.Arg404Phefs*4 and p.Tyr405Glufs*28, respectively for transcripts Δ8, Δ8q, and ▾8q (Fig. 4). The nomenclature of the c.1211+2T>G variant at the RNA and protein level, following HGVS recommendations and taking into account the results of the minigene assay, is: r.[1042_1211del, 1206_1211del, 1211_1212ins1211+1_1211+11; 1211+2u>g], p.[Glu348Valfs*5, Arg404Phefs*4, Tyr405Glufs*28].

Fig.3

Ex vivo splicing assays in the context of the pCAS minigene carrying the c.1211+2T>G (paternal) or the c.3947-3insG (maternal) variants. A) Structure of pCAS2 minigene. The white arrow represents the CMV promoter, black arrows represent the primers used for the RT-PCR experiments, boxes represent exons and lines introns. Star indicates a fluorescent primer. B) RT-PCR products separated on agarose gel following the ex vivo splicing assay performed on SORL1 exon 8 WT and mutant (c.1211+2T>G), and SORL1 exon 29 WT and mutant (c.3947-3insG). We observe a unique band for both WT exons 8 and 29. Exon 8 mutant c.1211+2T>G and exon 29 mutant c.3947-3insG show three different bands. However, the upper band of the mutant exon 29 correponds to a heteroduplex as revealed by its absence after fluorescent RT-PCR experiments (see C). The unique band in the pCAS lane corresponds to exon A – exon B splicing. C) Fluorescent RT-PCR products were separated by capillary electrophoresis. Exon 8 WT shows one unique fragment corresponding to the natural splicing of exon 8. Mutant exon 8 (c.1211+2T>C) shows 3 different RT-PCR products. Cloning and sequencing revealed that they correspond to the skipping of exon 8 (Δ8) and the activation of two different cryptic sites, c.1206_1207(Δ8q) and c.(1211+12)_(1211+13) (▾8q), respectively, as summarized schematically on panel D. Exon 29 WT shows one main fragment corresponding to the natural splicing of exon 29. Mutant exon 29 (c.3947-3insG) shows two different fragments. Cloning and sequencing revealed that they correspond to the activation of a cryptic acceptor splice site in c.3947-44_3947-43 combined with the use of the canonical donor site (▾29p) or the activation of a donor cryptic site in c.3970_3971 (▾29p Δ29q), as summarized schematically on panel D. Note that two tiny peaks migrating approximately in the same position as ▾29p and ▾29p Δ29q are observed in the WT position (indicated by dotted arrows), that we could not clone. We hypothesize that they correspond to transcripts ▾29p and ▾29p Δ29q without the insertion of the G. D) Schematic representation of the natural and aberrant transcript are displayed. Black boxes indicate included exonic sequence, grey boxes indicate intronic retention, and white boxes indicate excluded exonic sequence. Solid lines between exons represent natural splicing events, dotted lines represent aberrant splicing events.

Fig.4

Transcripts and expected proteins products as interpreted in the context of the SORL1 gene carrying the c.1211+2T>G (paternal) or the c.3947-3insG (maternal) variants. A) The paternal variant c.1211+2T>G is located in intron 8 and belongs to the canonical splicing donor site. The variant leads to the total loss of the canonical donor site and the generation of three different aberrant transcripts as shown in fluorescent RT-PCR experiments (see Fig. 3C), all resulting in a frameshift: one leading to the skipping of exon 8 with aberrant exon 7 - exon 9 junction (A, lower left part), one using a cryptic donor site in position c.1206_1207 leading to the loss of 5 bp of the exon 8 (lower middle part), and one using another cryptic donor site located in intron 8 in position c.1211+12_1211+13 (lower right part). These three aberrant transcripts would generate, if translated, 3 different truncated SORL1 proteins: p.Glu348Valfs*5, p.Arg404Phefs*4, and p.Tyr405Glufs*28, respectively. B) The maternal variant c.3947-3insG is located in intron 28, close the canonical acceptor site. The variant leads to the total loss of the canonical acceptor site and the generation of 2 different aberrant transcripts as shown in fluorescent RT-PCR experiments (see Fig. 3C), both resulting in a frameshift: both use a cryptic acceptor site located in intron 28 in position c.3947-44_3947-43, resulting in the inclusion of these 43 bases pairs plus the inserted G in cDNA (grey boxes and G colored in red); one of them uses the canonical donor site (lower left part), the other one uses a cryptic donor site within exon 29 (c.3970_3971, lower right part), resulting in a loss of 109 base pairs of exon 29 in cDNA. These two aberrant transcripts would generate, if translated, two different truncated SORL1 proteins: p.Gln1317Trpfs*39 and p.Gln1317Trpfs*39, respectively (same short nomenclature but the coding sequence is distinct).

The pCAS minigene containing WT exon 29 mainly produced the transcript with the normal exon inclusion at the expected size (366 nt), as determined by fluorescent RT-PCR and sequencing analyses (Fig. 3C and D, Supplementary Figure 1), whereas the construct containing the c.3947-3insG variant did not generate the latter normal transcript. Instead, two fragments migrating at ∼300 and ∼410 nt, respectively, were detected. Cloning and sequencing them revealed that they both contained the retention of the last 44 nt of intron 28 (taking into account the insertion of a G at position c.3947-3), consequence of the use of the cryptic intronic 3’ splice site. The longest product (410 nt) used the canonical 5’ splice site of exon 29 (transcript ▾29p), whereas the shortest fragment used a cryptic exonic 5’ splice site located at c.3970_3971 position, resulting in a deletion of 109 nt of exon 29 (transcript▾29pΔ29q). Transposing these minigene data into the physiological context of SORL1 transcript, the aberrant transcripts ▾29p and ▾29pΔ29q would both lead to the introduction of a premature stop codon, p.Gln1317Trpfs*39 (Fig. 4). The nomenclature of the c.3947-3insG variant at the RNA and protein level is: r.[3946_3947ins3947-43_3947-1; 3947-3insg, 3946_3947ins3947-43_3947-1; 3947-3insg; 3970_4078del], p.[Gln1317Trpfs*39, Gln1317Trpfs*39]. In both aberrant transcripts, the premature stop codon mapped to exon 29 or exon 30 coding sequence, confirming that whether or not the variant c.4265A>G, p.(N1422S) has an effect on exon 31 splicing, it would occur downstream of the premature stop codons in both aberrant transcripts created by the c.3947-3insG variant in the same allele. Of note, two tiny peaks migrating approximately at the same sizes as the two aberrant transcripts ▾29p and ▾29pΔ29q were also detected by fluorescent RT-PCR in the WT condition but we could not clone them to confirm the hypothesis being that they actually correspond to the same aberrant transcripts without the insertion of the G.

Finally, the two variants c.1211+2T>G and c.3947-3insG located in trans in this patient resulted in a total loss of any in frame transcript in the pCAS minigene context, resulting in a bi-allelic loss of function as interpreted in the SORL1 context.

DISCUSSION

By whole exome sequencing, we previously identified 9 patients with EOAD and carrying one PTV [6]. Among them, we show here that one also carries another PTV in the other allele, not annotated as such because falling outside the boundaries used in most of the exome case-controls studies for canonical splice sites. Both variants are predicted to result in NMD, as ascertained previously for other PTV [6 , 14]. This is therefore the first description of SORL1 bi-allelic loss of function in human, to our knowledge.

Interestingly, both parents developed dementia. Three generations were affected by dementia in the paternal lineage which was suggestive of autosomal dominant EOAD—defined by the presence of at least 2 patients from 2 generations with an age of onset before 66 [21]. The mother was affected by late onset AD and censoring effect was noticed in her own parents. SORL1 rare missense damaging variants or PTVs were initially identified in 7 probands from families with a pedigree suggesting autosomal dominant inheritance and without any pathogenic PSEN1, PSEN2, or APP variant [14]. However, the lack or paucity of segregation data in previously published families still precludes any conclusion regarding the role of SORL1 rare variants in a Mendelian hypothesis [6 , 23]. The effect of SORL1 rare missense damaging and PTVs has been assessed in a case-control setting by our group and others [6–8 , 24]. High ORs together with the absence of PTV in controls suggested a moderate to high effect on disease risk, with higher ORs and genome wide significance (p < 2.5 10^–6) achieved for familial EOAD [6]. Interestingly, in the gnomAD database gathering WES or WGS data from 138,642 individuals (http://gnomad.broadinstitute.org/) [17], 69 canonical splice site, nonsense, or frameshift variants are reported after exclusion of the low confidence PTVs, with a total allele count of 96, the allele count of the most recurrent rare SORL1 PTV being 10/122,924. Some of the carriers could be affected by AD as this status was not ascertained in gnomAD, or could develop AD later, or such variants could basically be associated with incomplete penetrance, which is equivalent as being considered as a strong risk factor. Interestingly, in gnomAD, there is no homozygous PTV carrier. Of note, the hypothesis that two PTV could be carried by a single individual in a compound heterozygous state cannot be assessed from gnomAD publicly available data. Assuming that all PTVs are carried by different individuals, the frequency of PTV carriers would be 6.9×10^–4 in gnomAD. This frequency seems to be consistent with the absence of PTV in controls from AD case controls series with sample sizes of controls in the ranges of 1,000–2,000 or less [6–8 , 24].

In Family ROU-0055, segregation analysis showed that c.3947-3insG and c.4265A>G, p.(N1422S) are inherited from the proband’s mother and that the c.1211+2T>G is in trans, in the second, paternal allele. Furthermore, in vitro analysis of splicing in minigene assays showed an introduction of premature stop codons in every transcript resulting from both intronic variants. If same splicing defects occur in neurons and if NMD is complete, patient 001 would have a virtually total loss of function of SORL1, i.e., the first human SORL1 KO. This is compatible with mouse models which are also viable [25]. Given the pedigrees and the ages at onset of other SORL1 PTV carriers at the heterozygous state (Supplementary Table 1), one could have expected a dramatic effect of such haploinsufficiency on disease onset. Although ages of onset could not be determined with precision in parents, it seems that the proband’s age at onset is earlier than his parents’, even if taking into account the determined and inferred APOE genotypes, but it remains far from the average age at onset in PSEN1 mutation carriers, evaluated around 44.4 years in our series [26].

Of note, we identified variants that introduce premature stop codons in the mRNA. These transcripts are predicted to be degraded by NMD. However, a proportion of the aberrant transcripts might escape this system—as previously observed with other SORL1 PTV [6]—and hence lead to the production of a truncated protein. If this abnormal protein is not degraded, it might retain part of its function. This is, however, probably not the case for the c.1211+2T>G variant which introduces a premature stop codon within the sequence encoding the VPS10 domain, which has been shown to bind Aβ [13]. In addition, the LDLR-class A repeat domain, mapping more in the 3’ region, would also be missing. The latter domain has been shown to bind AβPP [12]. We can therefore conclude that whether or not the transcripts resulting from aberrant splicing of the allele carrying the c.1211+2T>G variant exhibit full NMD, this variant is considered as a loss-of-function variant regarding AβPP and Aβ. However, regarding the c.3947-3insG variant introducing a premature stop codon in the LDLR-class A repeats domain, we cannot exclude that a partial protein function could remain if the mRNA is not totally degraded by NMD.

Here, we focused on rare variants (MAF <1%). We cannot exclude that low-frequency or common variants might affect splicing and result in a certain degree of loss of function. Given the PTV statistics in large databases such as gnomAD, we do not expect variants with a strong effect on splicing to be common, but variants with an intermediate effect could contribute to the risk of AD.

In conclusion, this is the first report of a human KO for SORL1, carrying PTVs in a compound heterozygous state. The normal neurodevelopment of this patient and aging during adulthood and the absence of a dramatic effect on disease onset suggest that SORL1 is not required for normal life until adulthood.

Footnotes

ACKNOWLEDGMENTS

This study was funded by grants from the Clinical Research Hospital Program from the French Ministry of Health (GMAJ, PHRC 2008/067), the CNR-MAJ, and the JPND PERADES. This work was also supported by the French Association Nationale de la Recherche et de la Technologie (ANRT, CIFRE PhD fellowship to H.T.), the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (Gefluc), and by the OpenHealth Institute.

This study was cosupported by Inserm, European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF).

The sponsors of the study had no role in study design, data collection, analysis and interpretation, or writing of the manuscript.

We are grateful to Séverine Jourdain for her technical help with blood samples.

Authors’ disclosures available online ().

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

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