23 Nov 2022

When digging for the maximum number of needles, it helps to start with a massive haystack. That strategy worked to pin rare variants in two novel genes to AD risk. Published November 21 in Nature Genetics, researchers led by Henne Holstege and Marc Hulsman of the VU University in Amsterdam, Gaël Nicolas of Normandie University in Rouen, France, and Jean-Charles Lambert of the Institut Pasteur de Lille in France harmonized exome sequencing data from consortia in North America and Europe to hunt for rare, yet impactful, genetic variants that influence AD risk. From the depths of more than 32,000 exomes, the researchers plucked some 7 million variants predicted to hobble expression or function of different genes. Among those, they tied variants in five—SORL1, TREM2, ABCA7, ATP8B4, and ABCA1—to AD risk. Coding variants in the latter two genes, which both encode proteins involved in lipid handling in microglia, had not been linked to AD risk before. However, common, less-harmful variants near all five genes had previously cropped up in genome-wide association studies. Together, the findings bolster support for the central roles of APP processing, lipid handling, and microglial function in AD.

Harmonization of European and American datasets yielded 32,558 exomes.
Rare variants in SORL1, TREM2, ABCA7, ATP8B4, and ABCA1 tied to AD risk.
The new ones—ATP8B4 and ABCA1—are involved in lipid handling in microglia.

Alzforum covered the bulk of the findings when they were presented at the Alzheimer’s Association International Conference in 2020, and also posted on medRxiv (Jul 2020 conference coverage). At that time, they had analyzed exomes from 12,652 AD cases and 8,693 controls, pulling out significant associations with AD risk in damaging variants within the SORL1, TREM2, ABCA7, and ATP8B4 genes. Variants in a handful of other genes—ADAM10, ABCA1, ORC6, CBX3, PRSS3, B3GNT4, and SRC—nearly missed significance in that initial analysis.

The published findings added exomes of 3,384 cases and 7,829 controls, which served to confirm the original findings and, when combined with the original dataset, to ramp up the total sample size to 16,036 AD cases and 16,522 controls. From this “mega-sample” of exomes, co-first authors Holstege, Marc Hulsman of Amsterdam University Medical Center, and Camille Charbonnier in Normandie found that in SORL1, TREM2, ABCA7, ATP8B4, and ABCA1, rare variants predicted to compromise gene expression or function significantly upped AD risk. Deleterious variants in the ADAM10 gene, which encodes α-secretase, nearly missed significance, likely because they were extremely rare and the analysis lacked sufficient power.

With the exception of ATP8B4, variants near all of these genes have been linked to AD risk in GWAS, which typically identifies common, less-harmful variants that land within noncoding regions near multiple genes. This motivated the researchers to focus on rare variants among the 75 previously reported GWAS hits. Four—RIN3, CLU, ZCWPW1, and ACE—harbored rare, deleterious coding variants, which were suggestively tied to AD risk in their analysis. The findings suggest that these genes, and not others at the same locus, could be the drivers of AD risk identified in GWAS.

The two novel risk genes identified in this study play central roles in lipid metabolism. As discussed in prior conference coverage, ATP8B4 is a transporter that ferries phospholipids across the cell membrane, and in the brain is predominantly expressed by microglia. ABCA1 also encodes a phospholipid transporter. It lipidates, among others, ApoE. When that fails, the apolipoprotein ramps up Aβ aggregation.—Jessica Shugart

Comments

- Dmitry Prokopenko
  Massachusetts General Hospital, Harvard Medical School
- Rudy Tanzi
  Massachusetts General Hospital
- Posted: 28 Nov 2022
Using multiple whole-exome sequencing (WES) datasets on early and late-onset Alzheimer’s disease patients and controls from several consortia, Holstege and colleagues assessed the burden of predicted, rare, damaging variants in exomes from roughly 32,000 subjects. The authors confirmed previously identified rare variant signals in the known AD genes: SORL1, TREM2, and ABCA7. They also report novel rare-variant driven signals in the known AD gene, ABCA1, as well as novel signals in the gene ATP8B4. This study also found ADAM10 to exhibit a suggestive rare-variant driven signal. This finding agrees with our previous studies showing co-segregation of two, rare, highly penetrant pathogenic (loss-of-function) mutations in the prodomain of ADAM10 with AD in late-onset AD families (Kim et al., 2009; Suh et al., 2013). Both ADAM10 mutations reduced α-secretase cleavage of APP by more than 60 percent.

Four other known AD GWAS loci (RIN3, CLU, ZCWPW1, and ACE) were also mapped to exonic burden signals. This suggests that at the SLC24A4/RIN3 AD GWAS locus, RIN3 is the more likely AD candidate gene. This agrees with our recent study demonstrating that β-secretase cleavage of APP, and Aβ generation, are regulated by the interaction of RIN3 with the neuronal form of BIN1, encoded by another AD GWAS gene (Bhattacharyya et al., 2022). One of the most insightful findings in this study was that only nine out of 75 known GWAS-validated AD loci tested could be mapped to loss-of-function, or rare damaging variants, in exons. Of course, for those AD loci deemed to harbor exonic loss-of-function or rare damaging variants, functional studies of specific mutations will be necessary to validate these findings in the future.

Overall, this study nicely adds to the growing literature of whole-exome and whole-genome sequencing datasets to search AD-associated rare variants in functionally relevant genomic regions in exons and beyond. For example, we and others have previously used different grouping strategies to identify more than a dozen novel rare-variant driven AD associations. This includes spatial clustering of rare variants based on their proximity along the genome, nonoverlapping consecutive sets of rare variants, and a protein structure-based approach (Prokopenko et al., 2021; Prokopenko et al., 2022; Jin et al., 2022).

The authors are to be congratulated for publishing such a comprehensive and well-executed WES-based study. This, and prior studies searching for AD-associated rare genomic variants, clearly shows that more effort is warranted to increase the statistical power to detect additional rare variant associations by both gathering significantly larger WES and WGS AD datasets for systematic analysis, and by performing hypothesis-driven studies with additional biological validation. With the ongoing expansion of AD WGS and WES datasets, it would be interesting to see more of such analyses in genomic regions beyond exons and stratified by population. It is also important to go beyond new gene identification by understanding how novel AD gene variants contribute to AD pathogenesis. The latter will require concerted efforts by the AD research community to begin testing specific functional variants and mutations in the AD loci that have been implicated in this and previous studies.

References:

Kim M, Suh J, Romano D, Truong MH, Mullin K, Hooli B, Norton D, Tesco G, Elliott K, Wagner SL, Moir RD, Becker KD, Tanzi RE. Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate {alpha}-secretase activity. Hum Mol Genet. 2009 Oct 15;18(20):3987-96. PubMed.

Suh J, Choi SH, Romano DM, Gannon MA, Lesinski AN, Kim DY, Tanzi RE. ADAM10 Missense Mutations Potentiate β-Amyloid Accumulation by Impairing Prodomain Chaperone Function. Neuron. 2013 Oct 16;80(2):385-401. PubMed.

Bhattacharyya R, Teves CA, Long A, Hofert M, Tanzi RE. The neuronal-specific isoform of BIN1 regulates β-secretase cleavage of APP and Aβ generation in a RIN3-dependent manner. Sci Rep. 2022 Mar 3;12(1):3486. PubMed. Correction.

Prokopenko D, Morgan SL, Mullin K, Hofmann O, Chapman B, Kirchner R, Alzheimer's Disease Neuroimaging Initiative (ADNI), Amberkar S, Wohlers I, Lange C, Hide W, Bertram L, Tanzi RE. Whole-genome sequencing reveals new Alzheimer's disease-associated rare variants in loci related to synaptic function and neuronal development. Alzheimers Dement. 2021 Sep;17(9):1509-1527. Epub 2021 Apr 2 PubMed.

Prokopenko D, Lee S, Hecker J, Mullin K, Morgan S, Katsumata Y, Alzheimer’s Disease Neuroimaging Initiative (ADNI), Weiner MW, Fardo DW, Laird N, Bertram L, Hide W, Lange C, Tanzi RE. Region-based analysis of rare genomic variants in whole-genome sequencing datasets reveal two novel Alzheimer's disease-associated genes: DTNB and DLG2. Mol Psychiatry. 2022 Apr;27(4):1963-1969. Epub 2022 Mar 4 PubMed.

Jin B, Capra JA, Benchek P, Wheeler N, Naj AC, Hamilton-Nelson KL, Farrell JJ, Leung YY, Kunkle B, Vadarajan B, Schellenberg GD, Mayeux R, Wang LS, Farrer LA, Pericak-Vance MA, Martin ER, Haines JL, Crawford DC, Bush WS. An association test of the spatial distribution of rare missense variants within protein structures identifies Alzheimer's disease-related patterns. Genome Res. 2022 Apr;32(4):778-790. Epub 2022 Feb 24 PubMed.

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- Iliya Lefterov
  University of Pittsburgh
- Radosveta Koldamova
  University of Pittsburgh
- Posted: 08 Dec 2022
The most recent quest for culprits of late-onset AD reveals well-known guardians of felicitous intracellular and transmembrane lipid transport (again).

The results of this study provide strong evidence that "damaging" genetic variants of genes coding for ATP binding phospholipids and cholesterol transporters ABCA1 and ABCA7 are significant risk of Alzheimer's disease. ATP8B4, another gene of comparable significance, codes for an ATPase transporter involved in phospholipids and cholesterol transport at the cell membrane. With the addition of APOE allelic variation (not included in this study), it is clear that dysfunctional proteins involved in cholesterol and phospholipids transport, and brain lipid metabolism, constitute the major risk of AD. While a direct involvement in APP processing for the proteins coded by the above genes has not been demonstrated so far, multiple studies at molecular, cellular, organism, and population levels have provided clear evidence of their possible role in AD pathogenesis. SORL1 codes for a transporter, too: the protein is involved in endocytosis and protein sorting. Mutations in this gene may be (not necessarily) associated with Alzheimer's disease.

A role of ABCA1 in AD was suggested more than 15 years ago. Seminal studies in APP-expressing mice later confirmed that lack of ABCA1 dramatically influences and aggravates the AD-like phenotype (Koldamova et al., 2005; Wahrle et al., 2005; Hirsch-Reinshagen et al., 2005). Subsequent reports demonstrated that effects of ABCA1 are translated in human APOE-isoform-dependent manner. In many of the studies the effects of ABCA1 were explained, or suggested, because of a dysfunctional LXR/RXR-ABCA1-APOE/APOA-I regulatory axis (Koldamova et al., 2005; Zelcer et al., 2007).

The results of this incredibly difficult to conduct study, which must have been made possible by the aggregation of enormous computational power and data provided by dozens of AD centers, hospitals, biostatistics, epidemiology and genetics departments, are an "observation of a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk." For the last 30 or so years hundreds of GWAS provided credible observations of significant associations of tens of common or rare gene variants, even before the concept of damaging variants had been defined. At the same time, however, the impact of molecular, cellular, and clinicopathological studies in the overall understanding of the role of a gene in AD pathogenesis cannot be underestimated. While there are many of those, the rare functional variant of ABCA1 is a good example. We are aware of two notable examples of ABCA1 mutations highly relevant to our understanding of its association to AD risk and AD pathogenesis.

The N935S mutation was identified in a patient with extremely low levels of HDL, but without accelerated development of premature atherosclerosis and with signs of severe dementia and amyloid deposition in the brain at age of 60. The second example is a compound heterozygous mutation (D1099Y and F2009S) identified in a subject with severe HDL-cholesterol deficiency. The patient had no history or clinical manifestation of coronary artery disease and no other cardiovascular disease risk factors, except for low HDL cholesterol. There were no clinical signs of Tangier Disease either. The patient developed and died of complications related to cerebral amyloid angiopathy (CAA). It is worth mentioning that vascular amyloid deposits are integral part of brain pathology observed in ABC1-deficient mice expressing mutant human APP. These two examples point to the significance of ABCA1 functional variation, which can be associated with AD risk, most probably operating through HDL cholesterol levels, although other mechanisms influencing APP processing cannot be excluded. With ABCA1, it is easy to make the story more complicated. In 2014 an Australian group identified low frequency, non-synonymous rare ABCA1 variants in control individuals, but not in AD cases (Lupton et al., 2014). The interpretation of the results, according to the authors, was suggestive of a protective effect. Importantly, the number of non-synonymous alleles of the previously identified rare variant E1172D, known to be associated with very high HDL-C levels, was more than twice as high in control as in case samples.

The authors of this study conclude that the burden of damaging ABCA1 variants is concentrated in younger patients and that the AD-association is mainly driven by variants that are extremely rare, but also by more common variants, and they provide as an example N1800H mutation. N1800H is a pathogenic, rare ABCA1 variant associated with cardiovascular disease due to low levels of HDL-C. There is no data in the article showing the level of HDL-C in AD patients or control carriers of damaging ABCA1, ABCA7 or ATP8B4 variants. Such a correlation might be an interesting (forgotten one maybe) and worth pursuing in future studies using the huge database already available.

This study, with no doubt, provides valuable information for molecular and cell biologists and geneticists who try to understand the role of lipid and cholesterol transporters in AD pathogenesis. An important question, however, remains unanswered: Do GWAS + exome sequencing studies provide more meaningful information to clinicians than the hundreds of GWAS studies conducted during the last 30 or so years?

References:

Koldamova R, Staufenbiel M, Lefterov I. Lack of ABCA1 considerably decreases brain ApoE level and increases amyloid deposition in APP23 mice. J Biol Chem. 2005 Dec 30;280(52):43224-35. PubMed.

Wahrle SE, Jiang H, Parsadanian M, Hartman RE, Bales KR, Paul SM, Holtzman DM. Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease. J Biol Chem. 2005 Dec 30;280(52):43236-42. PubMed.

Hirsch-Reinshagen V, Maia LF, Burgess BL, Blain JF, Naus KE, McIsaac SA, Parkinson PF, Chan JY, Tansley GH, Hayden MR, Poirier J, Van Nostrand W, Wellington CL. The absence of ABCA1 decreases soluble ApoE levels but does not diminish amyloid deposition in two murine models of Alzheimer disease. J Biol Chem. 2005 Dec 30;280(52):43243-56. PubMed.

Koldamova RP, Lefterov IM, Staufenbiel M, Wolfe D, Huang S, Glorioso JC, Walter M, Roth MG, Lazo JS. The liver X receptor ligand T0901317 decreases amyloid beta production in vitro and in a mouse model of Alzheimer's disease. J Biol Chem. 2005 Feb 11;280(6):4079-88. PubMed.

Zelcer N, Khanlou N, Clare R, Jiang Q, Reed-Geaghan EG, Landreth GE, Vinters HV, Tontonoz P. Attenuation of neuroinflammation and Alzheimer's disease pathology by liver x receptors. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10601-6. PubMed.

Lupton MK, Proitsi P, Lin K, Hamilton G, Daniilidou M, Tsolaki M, Powell JF. The Role of ABCA1 Gene Sequence Variants on Risk of Alzheimer's Disease. J Alzheimers Dis. 2013 Sep 30; PubMed.

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