Bruno A. Benitez

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimers disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

GWAS of Cerebrospinal Fluid Tau Levels Identifies Risk Variants for Alzheimer’s Disease

Cerebrospinal fluid (CSF) tau, tau phosphorylated at threonine 181 (ptau), and Aβ₄₂ are established biomarkers for Alzheimers disease (AD) and have been used as quantitative traits for genetic analyses. We performed the largest genome-wide association study for cerebrospinal fluid (CSF) tau/ptau levels published to date (n = 1,269), identifying three genome-wide significant loci for CSF tau and ptau: rs9877502 (p = 4.89 × 10⁻⁹ for tau) located at 3q28 between GEMC1 and OSTN, rs514716 (p = 1.07 × 10⁻⁸ and p = 3.22 × 10⁻⁹ for tau and ptau, respectively), located at 9p24.2 within GLIS3 and rs6922617 (p = 3.58 × 10⁻⁸ for CSF ptau) at 6p21.1 within the TREM gene cluster, a region recently reported to harbor rare variants that increase AD risk. In independent data sets, rs9877502 showed a strong association with risk for AD, tangle pathology, and global cognitive decline (p = 2.67 × 10⁻⁴, 0.039, 4.86 × 10⁻⁵, respectively) illustrating how this endophenotype-based approach can be used to identify new AD risk loci.

TREM2 Variant p.R47H as a Risk Factor for Sporadic Amyotrophic Lateral Sclerosis

IMPORTANCE Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which microglia play a significant and active role. Recently, a rare missense variant (p.R47H) in the microglial activating gene TREM2 was found to increase the risk of several neurodegenerative diseases, including Alzheimer disease. Whether the p.R47H variant is a risk factor for ALS is not known. OBJECTIVES To determine whether p.R47H (rs75932628) in TREM2 is a risk factor for ALS and assess whether TREM2 expression is dysregulated in disease. DESIGN, SETTING, AND PARTICIPANTS Samples of DNA from 923 individuals with sporadic ALS and 1854 healthy control individuals self-reported as non-Hispanic white were collected from ALS clinics in the United States and genotyped for the p.R47H variant in TREM2. Clinical data were obtained on ALS participants for genotype/phenotype correlations. Expression of TREM2 was measured by quantitative polymerase chain reaction and compared in spinal cord samples from 18 autopsied patients with ALS and 12 neurologically healthy controls, as well as from wild-type and transgenic SOD1G93A mice. MAIN OUTCOMES AND MEASURES Minor allele frequency of rs75932628 and relative expression of TREM2. RESULTS The TREM2 variant p.R47H was more common in patients with ALS than in the controls and is therefore a significant risk factor for ALS (odds ratio, 2.40; 95% CI, 1.29-4.15; P = 4.1×10-3). Furthermore, TREM2 expression was increased in spinal cord samples from ALS patients and SOD1G93A mice (P = 2.8×10-4 and P = 2.8×10-9, respectively), confirming dysregulated TREM2 in disease. Expression of TREM2 in the human spinal cord was negatively correlated with survival (P = .04) but not with other phenotypic aspects of disease. CONCLUSIONS AND RELEVANCE This study demonstrates that the TREM2 p.R47H variant is a potent risk factor for sporadic ALS. To our knowledge, these findings identify the first genetic influence on neuroinflammation in ALS and highlight the TREM2 signaling pathway as a therapeutic target in ALS and other neurodegenerative diseases.

TREM2 is associated with the risk of Alzheimer's disease in Spanish population

Two recent studies have reported the association of rs75932628-T in the TREM2 gene with the risk for Alzheimers disease (AD). Rs75932628-T is a rare nonsynonymous variant (p.R47H) that confers a high risk of AD with an effect size similar to that of the APOE ɛ4 allele. However, this association has not been replicated in any independent studies to date. The allelic frequency of rs75932628 varies according to the population from 0.02% to 0.63% among healthy controls. In an attempt to replicate the association between rs75932628-T and AD risk, we genotyped rs75932628 in a cohort of 504 AD subjects and 550 healthy controls from a Spanish population. Rs75932628-T showed a minor allele frequency of 0.3% among this cohort. Interestingly, in our study, rs75932628-T was found exclusively in 1.4% of AD cases (7/504), including 4 early-onset AD cases, and in none of the controls (n = 0/550). Here, we report the first positive replication study in a Spanish population and confirm that TREM2 rs75932628-T is associated with the risk for AD.

Missense variant in TREML2 protects against Alzheimer's disease

TREM and TREM-like receptors are a structurally similar protein family encoded by genes clustered on chromosome 6p21.11. Recent studies have identified a rare coding variant (p.R47H) in TREM2 that confers a high risk for Alzheimers disease (AD). In addition, common single nucleotide polymorphisms in this genomic region are associated with cerebrospinal fluid biomarkers for AD and a common intergenic variant found near the TREML2 gene has been identified to be protective for AD. However, little is known about the functional variant underlying the latter association or its relationship with the p.R47H. Here, we report comprehensive analyses using whole-exome sequencing data, cerebrospinal fluid biomarker analyses, meta-analyses (16,254 cases and 20,052 controls) and cell-based functional studies to support the role of the TREML2 coding missense variant p.S144G (rs3747742) as a potential driver of the meta-analysis AD-associated genome-wide association studies signal. Additionally, we demonstrate that the protective role of TREML2 in AD is independent of the role of TREM2 gene as a risk factor for AD.

Exome-Sequencing Confirms DNAJC5 Mutations as Cause of Adult Neuronal Ceroid-Lipofuscinosis

We performed whole-exome sequencing in two autopsy-confirmed cases and an elderly unaffected control from a multigenerational family with autosomal dominant neuronal ceroid lipofuscinosis (ANCL). A novel single-nucleotide variation (c.344T>G) in the DNAJC5 gene was identified. Mutational screening in an independent family with autosomal dominant ANCL found an in-frame single codon deletion (c.346_348 delCTC) resulting in a deletion of p.Leu116del. These variants fulfill all genetic criteria for disease-causing mutations: they are found in unrelated families with the same disease, exhibit complete segregation between the mutation and the disease, and are absent in healthy controls. In addition, the associated amino acid substitutions are located in evolutionarily highly conserved residues and are predicted to functionally affect the encoded protein (CSPα). The mutations are located in a cysteine-string domain, which is required for membrane targeting/binding, palmitoylation, and oligomerization of CSPα. We performed a comprehensive in silico analysis of the functional and structural impact of both mutations on CSPα. We found that these mutations dramatically decrease the affinity of CSPα for the membrane. We did not identify any significant effect on palmitoylation status of CSPα. However, a reduction of CSPα membrane affinity may change its palmitoylation and affect proper intracellular sorting. We confirm that CSPα has a strong intrinsic aggregation propensity; however, it is not modified by the mutations. A complementary disease-network analysis suggests a potential interaction with other NCLs genes/pathways. This is the first replication study of the identification of DNAJC5 as the disease-causing gene for autosomal dominant ANCL. The identification of the novel gene in ANCL will allow us to gain a better understanding of the pathological mechanism of ANCLs and constitutes a great advance toward the development of new molecular diagnostic tests and may lead to the development of potential therapies.

C9orf72 hexanucleotide repeat expansions in clinical Alzheimer disease.

IMPORTANCE Hexanucleotide repeat expansions in the chromosome 9 open reading frame 72 (C9orf72) gene underlie a significant fraction of frontotemporal dementia and amyotrophic lateral sclerosis. OBJECTIVE To investigate the frequency of C9orf72 repeat expansions in clinically diagnosed late-onset Alzheimer disease (AD). DESIGN, SETTING, AND PATIENTS This case-control study genotyped the C9orf72 repeat expansion in 872 unrelated familial AD cases and 888 control subjects recruited as part of the National Institute on Aging Late-Onset Alzheimer Disease Family Study cohort, a multisite collaboration studying 1000 families with 2 or more individuals clinically diagnosed as having late-onset AD. MAIN OUTCOMES AND MEASURES We determined the presence or absence of the C9orf72 repeat expansion by repeat-primed polymerase chain reaction, the length of the longest nonexpanded allele, segregation of the genotype with disease, and clinical features of repeat expansion carriers. RESULTS Three families showed large C9orf72 hexanucleotide repeat expansions. Two additional families carried more than 30 repeats. Segregation with disease could be demonstrated in 3 families. One affected expansion carrier had neuropathology compatible with AD. In the National Institute on Aging Late-Onset Alzheimer Disease Family Study series, the C9orf72 repeat expansions constituted the second most common pathogenic mutation, just behind the PSEN1 A79V mutation, highlighting the heterogeneity of clinical presentations associated with repeat expansions. CONCLUSIONS AND RELEVANCE C9orf72 repeat expansions explain a small proportion of patients with a clinical presentation indistinguishable from AD, and they highlight the necessity of screening frontotemporal dementia genes in clinical AD cases with strong family history.

Pooled-DNA sequencing identifies novel causative variants in PSEN1, GRN and MAPT in a clinical early-onset and familial Alzheimer's disease Ibero-American cohort

IntroductionSome familial Alzheimers disease (AD) cases are caused by rare and highly-penetrant mutations in APP, PSEN1, and PSEN2. Mutations in GRN and MAPT, two genes associated with frontotemporal dementia (FTD), have been found in clinically diagnosed AD cases. Due to the dramatic developments in next-generation sequencing (NGS), high-throughput sequencing of targeted genomic regions of the human genome in many individuals in a single run is now cheap and feasible. Recent findings favor the rare variant-common disease hypothesis by which the combination effects of rare variants could explain a large proportion of the heritability. We utilized NGS to identify rare and pathogenic variants in APP, PSEN1, PSEN2, GRN, and MAPT in an Ibero-American cohort.MethodsWe performed pooled-DNA sequencing of each exon and flanking sequences in APP, PSEN1, PSEN2, MAPT and GRN in 167 clinical and 5 autopsy-confirmed AD cases (15 familial early-onset, 136 sporadic early-onset and 16 familial late-onset) from Spain and Uruguay using NGS. Follow-up genotyping was used to validate variants. After genotyping additional controls, we performed segregation and functional analyses to determine the pathogenicity of validated variants.ResultsWe identified a novel G to T transition (g.38816G>T) in exon 6 of PSEN1 in a sporadic early-onset AD case, resulting in a previously described pathogenic p.L173F mutation. A pathogenic p.L392V mutation in exon 11 was found in one familial early-onset AD case. We also identified a novel CC insertion (g.10974_10975insCC) in exon 8 of GRN, which introduced a premature stop codon, resulting in nonsense-mediated mRNA decay. This GRN mutation was associated with lower GRN plasma levels, as previously reported for other GRN pathogenic mutations. We found two variants in MAPT (p.A152T, p.S318L) present only in three AD cases but not controls, suggesting that these variants could be risk factors for the disease.ConclusionsWe found pathogenic mutations in PSEN1, GRN and MAPT in 2.33% of the screened cases. This study suggests that pathogenic mutations or risk variants in MAPT and in GRN are as frequent in clinical AD cases as mutations in APP, PSEN1 and PSEN2, highlighting that pleiotropy of MAPT or GRN mutations can influence both FTD and AD phenotypic traits.

TREM2 is associated with increased risk for Alzheimer's disease in African Americans.

BackgroundTREM2 encodes for triggering receptor expressed on myeloid cells 2 and has rare, coding variants that associate with risk for late-onset Alzheimer’s disease (LOAD) in Caucasians of European and North-American origin. This study evaluated the role of TREM2 in LOAD risk in African-American (AA) subjects. We performed exonic sequencing and validation in two independent cohorts of >800 subjects. We selected six coding variants (p.R47H, p.R62H, p.D87N, p.E151K, p.W191X, and p.L211P) for case–control analyses in a total of 906 LOAD cases vs. 2,487 controls.ResultsWe identified significant LOAD risk association with p.L211P (p = 0.01, OR = 1.27, 95%CI = 1.05-1.54) and suggestive association with p.W191X (p = 0.08, OR = 1.35, 95%CI = 0.97-1.87). Conditional analysis suggests that p.L211P, which is in linkage disequilibrium with p.W191X, may be the stronger variant of the two, but does not rule out independent contribution of the latter. TREM2 p.L211P resides within the cytoplasmic domain and p.W191X is a stop-gain mutation within the shorter TREM-2V transcript. The coding variants within the extracellular domain of TREM2 previously shown to confer LOAD risk in Caucasians were extremely rare in our AA cohort and did not associate with LOAD risk.ConclusionsOur findings suggest that TREM2 coding variants also confer LOAD risk in AA, but implicate variants within different regions of the gene than those identified for Caucasian subjects. These results underscore the importance of investigating different ethnic populations for disease risk variant discovery, which may uncover allelic heterogeneity with potentially diverse mechanisms of action.

The PSEN1, p.E318G Variant Increases the Risk of Alzheimer's Disease in APOE-e4 Carriers

The primary constituents of plaques (Aβ42/Aβ40) and neurofibrillary tangles (tau and phosphorylated forms of tau [ptau]) are the current leading diagnostic and prognostic cerebrospinal fluid (CSF) biomarkers for AD. In this study, we performed deep sequencing of APP, PSEN1, PSEN2, GRN, APOE and MAPT genes in individuals with extreme CSF Aβ42, tau, or ptau levels. One known pathogenic mutation (PSEN1 p.A426P), four high-risk variants for AD (APOE p.L46P, MAPT p.A152T, PSEN2 p.R62H and p.R71W) and nine novel variants were identified. Surprisingly, a coding variant in PSEN1, p.E318G (rs17125721-G) exhibited a significant association with high CSF tau (p = 9.2×10−4) and ptau (p = 1.8×10−3) levels. The association of the p.E318G variant with Aβ deposition was observed in APOE-ε4 allele carriers. Furthermore, we found that in a large case-control series (n = 5,161) individuals who are APOE-ε4 carriers and carry the p.E318G variant are at a risk of developing AD (OR = 10.7, 95% CI = 4.7–24.6) that is similar to APOE-ε4 homozygous (OR = 9.9, 95% CI = 7.2.9–13.6), and double the risk for APOE-ε4 carriers that do not carry p.E318G (OR = 3.9, 95% CI = 3.4–4.4). The p.E318G variant is present in 5.3% (n = 30) of the families from a large clinical series of LOAD families (n = 565) and exhibited a higher frequency in familial LOAD (MAF = 2.5%) than in sporadic LOAD (MAF = 1.6%) (p = 0.02). Additionally, we found that in the presence of at least one APOE-ε4 allele, p.E318G is associated with more Aβ plaques and faster cognitive decline. We demonstrate that the effect of PSEN1, p.E318G on AD susceptibility is largely dependent on an interaction with APOE-ε4 and mediated by an increased burden of Aβ deposition.