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. Author manuscript; available in PMC: 2015 Oct 2.
Published in final edited form as: Nature. 2015 Apr 2;520(7545):E2–E3. doi: 10.1038/nature14038

PLD3-variants in population studies

Sven J van der Lee 1, Henne Holstege 2,3, Tsz Hang Wong 4, Johanna Jakobsdottir 5, Joshua C Bis 6, Vincent Chouraki 7,8, Jeroen GJ van Rooij 9, Megan L Grove 10, Albert V Smith 11,12, Najaf Amin 13, Seung-Hoan Choi 14,15, Alexa S Beiser 16,17, Melissa E Garcia 18, Wilfred FJ van IJcken 19, Yolande AL Pijnenburg 20, Eva Louwersheimer 21, Rutger WW Brouwer 22, Mirjam CGN van den Hout 23, Edwin Oole 24, Gudny Eirkisdottir 25, Daniel Levy 26,27,28, Jerome I Rotter 29, Valur Emilsson 30,31, Christopher J O’Donnell 32,33, Thor Aspelund 34,35, Andre G Uitterlinden 36,37, Lenore J Launer 38, Albert Hofman 39; ADNI*, Eric Boerwinkle 40,41, Bruce M Psaty 42,43,44,45, Anita L DeStefano 46,47, Philip Scheltens 48, Sudha Seshadri 49,50, John C van Swieten 51,52, Vilmundur Gudnason 53,54, Wiesje M van der Flier 55,56, M Arfan Ikram 57,58,59, Cornelia M van Duijn 60,#
PMCID: PMC4544703  NIHMSID: NIHMS714932  PMID: 25832410

Cruchaga et al.1 report that rare genetic variants in PLD3(phospholipase D3) are associated with increased Alzheimer’s disease(AD) risk. They showed that PLD3 is involved in amyloid-β precursor protein (APP) processing and overexpressed in brain tissue from AD patients. However, even the key variant PLD3-Val232Met, did not pass genome wide significance. This observation raises the question if the genetic association of PLD3 with AD replicates. We associated PLD3-Val232Met with AD in 3 large population-based studies and 3 case-control studies. In total, we meta-analyzed results from 1914 AD cases and 8021 controls of European descent. Additionally we searched for other coding PLD3-variants in sequence data of 1067 AD cases and 1553 controls.

Carrier frequencies of PLD3-Val232Met in controls ranged from 0.34-1.42%, consistent with 0-1.17% reported by Cruchaga et al.1 (Table 1). Likewise, the frequencies of PLD3-Val232Met in cases ranged from 0.66-2.19% compared to 0.7-2.6%.1 We note that the range of carrier frequencies overlaps between cases and controls, such that in some population based cohorts, the carrier frequency in controls (e.g., 1.28% in FHS) is higher than that of cases in other cohorts (e.g., 0.68% in AGES). Within each cohort frequencies of PLD3-Val232Met were higher in cases than controls (Table 1), but in none of the populations the case carrier frequency for PLD3-Val232Met was significantly increased. However, pooled analyses showed a 1.94 fold increased risk of AD of carriers compared to non-carriers (Odds Ratio [OR] 1.94, adjusted for age and sex, 95% confidence interval=1.05 to 3.57) that was marginally significant (p-value = 0.03)(Table 1). Of note, the crude ORs often differed considerably from the age and sex adjusted estimates. With the exception of ADNI, the ORs were higher after adjustment for age and sex, suggesting that many a-symptomatic carriers were relatively young compared to cases and age needs to be controlled for in the analyses as a putative confounder.

Table 1.

Association of PLD3-Val232Met with Alzheimer’s disease

Cohort Cases Controls Overall carrier frequency (%) Crude OR OR (95% CI) P-value
Carriers (N) Non-carriers (N) Carrier frequency (%) Carriers (N) Non-carriers (N) Carrier frequency (%)
AGES 1 145 0.68 12 2371 0.50 0.51 1.36 3.18 (0.17 - 58.73) 0.44
Dutch Alzheimer centers 3 451 0.66 4 609 0.65 0.66 1.01 1.55 (0.25 - 9.38) 0.64
GRIP 2 109 1.80 14 975 1.42 1.45 1.28 1.58 (0.25 - 9.91) 0.62
RS 6 470 1.26 23 2389 0.95 1.00 1.33 1.43 (0.51 - 4.03) 0.49
ADNI 7 492 1.40 1 292 0.34 1.01 4.15 2.94 (0.63 - 13.8) 0.17
FHS 5 223 2.19 17 1314 1.28 1.41 1.73 2.63 (0.71 - 9.70) 0.15

Combined 24 1890 1.25 71 7950 0.89 0.96 1.53 1.94 (1.05 - 3.57) 0.03

Odds Ratios(OR) and p-values from score tests are shown adjusted for age and sex, based on a logistic regression model. Crude ORs were calculated using carrier frequencies of cases and controls. Combined crude OR is the Mantel-Haenszel estimate of the pooled crude ORs(95% CI 0.91 to 2.57 and two-sided p-value=0.11). Age, Gene/Environment Susceptibility-Reykjavik Study(AGES). Framingham Heart Study(FHS) and Rotterdam Study(RS) where genotyped on the Illumina exome chip version 1.0. Genetic Research in Isolated Populations(GRIP) subjects were imputed. Dutch Alzheimer centers; encompass whole exome sequence data of AD cases from Amsterdam Dementia Cohort, Alzheimer Center Rotterdam MC and controls from RS(not genotyped on the exome chip). Alzheimer’s Disease Neuroimaging Initiative(ADNI) samples are whole genome sequenced.

We further associated other coding variants in PLD3 with AD and performed a gene-based test using sequence data from two studies encompassing 1067 AD cases and 1553 controls. We meta-analyzed results of whole genome sequence data of the ADNI study, 499 AD cases and 293 controls, with results of a combined cohort of 568 Dutch AD cases and 1260 Dutch controls. We observed 21 rare polymorphic coding variants and 1 splice site variant. Of the 20 observed PLD3-variants detected by Cruchaga et al.1, we observed 9 (S63G, P76A, V232M, N284S, C300Y, A442A, G452E, D447G and R488C). Five variants showed the same direction of effect as seen by Cruchaga et al.1. PLD3-A442A was one of the variants that showed a same direction of effect(Odds Ratio [OR] 1.24, 95% confidence interval = 0.74 to 2.06, p-value = 0.41). After correcting the p-value for multiple testing, none of the variants observed in our study conferred a significant increase in AD risk. Gene-based analysis also did not show significant association of PLD3-variants with AD risk (SKAT-O p-value = 0.61 and burden test OR 1.27 95% confidence interval [CI] =0.85 to 1.9, p-value = 0.24).

In conclusion, the carrier frequencies of PLD3-Val232Met in our data set are consistent with those reported by Cruchaga et al.1 and we showed a nominally significant association of PLD3-Val232Met with AD. This is in contrast to findings presented in companion papers by Heilmann et al., Lambert et al. and Hooli et al. However, in contrast to Cruchaga et al.1 we found no significant association of other PLD3-variants with AD in the single variant or gene based analyses. Therefore, in our analyses PLD3 does not yet meet the criteria proposed by MacArthur et al.2 to be implicated in AD. Hence, our data do not strongly support an important contribution of rare PLD3-variants in the etiology of AD. The most notable findings in our study are the need to control for age as a confounder in rare variant analyses and the high variability of the frequency of PLD3-Val232Met across populations. The latter finding highlights the need for careful matching of cases and controls for ethnic background when investigating rare variants.

Methods

RS3, FHS4 and AGES5 were genotyped on the Illumina exome chip6. Amsterdam Dementia cohort7, Alzheimer Center Erasmus MC and RS underwent whole exome sequencing at Center for Biomics, Rotterdam. RS exome sequence and exome chip data partially overlapped, genotypes were concordant and non-overlapping samples were used in PLD3-Val232Met analysis. As covariates gender and age at onset for AD cases or the date of last examination/censoring for cognitively healthy controls were used. GRIP8 was imputed using the Dutch specific reference panel(Imputation quality [Rsq]=0.74)9,10. Subjects aged below 55 were excluded. The R-package “seqMeta”(version seqMeta_1.4) was used for meta-analysis of single variant score test and gene-based test.

Contributor Information

Sven J. van der Lee, Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands

Henne Holstege, Alzheimer Center, Department of Neurology, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands; Department of Clinical Genetics, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands.

Tsz Hang Wong, Department of Neurology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.

Johanna Jakobsdottir, Icelandic Heart Association, Kopavogur, Iceland.

Joshua C. Bis, Cardiovascular Health Research Unit, University of Washington, Seattle, WA, USA

Vincent Chouraki, Framingham Heart Study, Framingham, MA, USA; Boston University School of Medicine, Boston, MA, USA.

Jeroen G.J. van Rooij, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands

Megan L. Grove, School of Public Health, Human Genetics Center, University of Texas Health Science Center at Houston, Houston, TX, USA

Albert V. Smith, Icelandic Heart Association, Kopavogur, Iceland Faculty of Medicine, University of Iceland, Reykjavik, Iceland.

Najaf Amin, Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands.

Seung-Hoan Choi, Framingham Heart Study, Framingham, MA, USA; Department of Biostatistics, Boston University School of Public Health, Boston, MA, USA.

Alexa S. Beiser, Framingham Heart Study, Framingham, MA, USA Department of Biostatistics, Boston University School of Public Health, Boston, MA, USA.

Melissa E. Garcia, Laboratory of Epidemiology, Demography, and Biometry, National Institute on Aging, Bethesda, MD, USA

Wilfred F.J. van IJcken, Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands

Yolande A.L. Pijnenburg, Alzheimer Center, Department of Neurology, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands

Eva Louwersheimer, Alzheimer Center, Department of Neurology, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands.

Rutger W.W. Brouwer, Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands

Mirjam C.G.N. van den Hout, Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands

Edwin Oole, Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands.

Gudny Eirkisdottir, Icelandic Heart Association, Kopavogur, Iceland.

Daniel Levy, Framingham Heart Study, Framingham, MA, USA; Boston University School of Medicine, Boston, MA, USA; Heart, Lung, and Blood Institute, Framingham, MA, USA.

Jerome I. Rotter, Institute for Translational Genomics and Population Sciences, Los Angeles BioMedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA

Valur Emilsson, Icelandic Heart Association, Kopavogur, Iceland; Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.

Christopher J. O’Donnell, Framingham Heart Study, Framingham, MA, USA Heart, Lung, and Blood Institute, Framingham, MA, USA.

Thor Aspelund, Icelandic Heart Association, Kopavogur, Iceland; Centre for Public Health, University of Iceland, Reykjavik, Iceland.

Andre G. Uitterlinden, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands Netherlands Consortium on Health Aging and National Genomics Initiative, Leiden, The Netherlands.

Lenore J. Launer, Laboratory of Epidemiology, Demography, and Biometry, National Institute on Aging, Bethesda, MD, USA

Albert Hofman, Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands.

Eric Boerwinkle, School of Public Health, Human Genetics Center, University of Texas Health Science Center at Houston, Houston, TX, USA; Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA.

Bruce M. Psaty, Cardiovascular Health Research Unit, University of Washington, Seattle, WA, USA Department of Epidemiology, University of Washington, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA; Group Health Research Institute, Seattle, WA, USA.

Anita L. DeStefano, Framingham Heart Study, Framingham, MA, USA Department of Biostatistics, Boston University School of Public Health, Boston, MA, USA.

Philip Scheltens, Alzheimer Center, Department of Neurology, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands.

Sudha Seshadri, Framingham Heart Study, Framingham, MA, USA; Boston University School of Medicine, Boston, MA, USA.

John C. van Swieten, Department of Neurology, Erasmus MC University Medical Center, Rotterdam, The Netherlands Alzheimer Center, Department of Neurology, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands.

Vilmundur Gudnason, Icelandic Heart Association, Kopavogur, Iceland; Faculty of Medicine, University of Iceland, Reykjavik, Iceland.

Wiesje M. van der Flier, Alzheimer Center, Department of Neurology, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands Department of Epidemiology & Biostatistics, VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam, The Netherlands.

M. Arfan Ikram, Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands; Department of Neurology, Erasmus MC University Medical Center, Rotterdam, The Netherlands; Departments of Radiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.

Cornelia M. van Duijn, Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands.

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