Novel and prevalent non-East Asian ALDH2 variants; Implications for global susceptibility to aldehydes' toxicity - PubMed (original) (raw)

doi: 10.1016/j.ebiom.2020.102753. Epub 2020 May 8.

Julio C B Ferreira 2, Amit U Joshi 1, Matthew C Stevens 1, Sin-Jin Li 3, Jade H-M Hsu 4, Rory Maclean 1, Nikolas D Ferreira 5, Pilar R Cervantes 6, Diana D Martinez 6, Fernando L Barrientos 6, Gibran H R Quintanares 6, Daria Mochly-Rosen 7

Affiliations

Novel and prevalent non-East Asian ALDH2 variants; Implications for global susceptibility to aldehydes' toxicity

Che-Hong Chen et al. EBioMedicine. 2020 May.

Erratum in

Expression of concern in

Abstract

Background: Aldehyde dehydrogenase 2 (ALDH2) catalyzes the detoxification of aliphatic aldehydes, including acetaldehyde. About 45% of Han Chinese (East Asians), accounting for 8% of humans, carry a single point mutation in ALDH2*2 (E504K) that leads to accumulation of toxic reactive aldehydes.

Methods: Sequencing of a small Mexican cohort and a search in the ExAC genomic database for additional ALDH2 variants common in various ethnic groups was set to identify missense variants. These were evaluated in vitro, and in cultured cells expressing these new and common variants.

Findings: In a cohort of Hispanic donors, we identified 2 novel mutations in ALDH2. Using the ExAC genomic database, we found these identified variants and at least three other ALDH2 variants with a single point mutation among Latino, African, South Asian, and Finnish ethnic groups, at a frequency of >5/1000. Although located in different parts of the ALDH2 molecule, these common ALDH2 mutants exhibited a significant reduction in activity compared with the wild type enzyme in vitro and in 3T3 cells overexpressing each of the variants, and a greater ethanol-induced toxicity. As Alda-1, previously identified activator, did not activate some of the new mutant ALDH2 enzymes, we continued the screen and identified Alda-64, which is effective in correcting the loss of activity in most of these new and common ALDH2 variants.

Interpretation: Since ~80% of the world population consumes ethanol and since acetaldehyde accumulation contributes to a variety of diseases, the identification of additional inactivating variants of ALDH2 in different ethnic groups may help develop new 'precision medicine' for carriers of these inactive ALDH2.

Keywords: ALDH2 deficiency; Alcohol toxicity; Alda-1 and -64; Health burden; Novel mutations.

Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.

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Conflict of interest statement

Declaration of Competing Interest Daria Mochly-Rosen and Che-Hong Chen hold patents related to Alda-1 activation of ALDH2*1 and ALDH2*2. One of the patents is licensed to Foresee Pharmaceuticals, a company that DM-R consults. However, these authors do not own stocks of the company and none of this research is supported by the company.

Figures

Fig 1

Fig. 1

Unstudied, high-frequency variants of ALDH2 have been extracted from the ExAC database (A). Sequence analysis from human samples showing examples of the Latino _ALDH2*_4 (P92T) and _ALDH2*_6 (V304M) variants. (B) All mutations identified in the Exome Aggregation Consortium (ExAC) database are shown on a linearized map of the ALDH2 protein structure. (C) Previously uncharacterized mutations in ALDH2 were identified using the ExAC database. Those variants with allele frequency percentages greater than 0.1% in a given ethnic population are shown in Table S1, along with the previously studied mutant E504K. Mutation numbering refers to the amino acid sequence of the mature protein with the 17-amino acid mitochondrial targeting sequence. Color code assignments are used consistently in the reporting of this work. Ethnicity assignments were made based off of available information in the ExAC database and might not represent all affected populations. (D) The mutated residues are shown with their respective color codes on the crystal structure (PDB:

1O05

) of the ALDH2 monomer (top) and tetramer (bottom, inset).

Fig 2

Fig. 2

Newly characterized ALDH2 mutants show decreased activity in vitro compared to wildtype (WT) enzyme. (A)ALDH2 activity of the recombinant mutants upon acetaldehyde treatment was measured using the conversion of NAD+ to NADH, without treatment (solid bars) and with the ALDH2 activators Alda-1 and Alda-64 (checkered and striped bars, respectively). n = 3–6; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _*** p_-value <0.001; _** p_-value <0.01; _* p_-value <0.05. (B) Fold change of ALDH2 activity improvement in each individual ALDH2 variants by Alda-1 and Alda-64. (C) Dehydrogenase activity measured in other ALDH isoforms after treatment with Alda-1 and Alda-64. n = 3; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _* p_-value <0.05. (D)ALDH2 activity measured in enzyme produced in a co-expression system of WT and ALDH2 variants to mimic heterozygous carriers. n = 3; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _** p_-value <0.01; _* p_-value <0.05.

Fig 3

Fig 3

Novel ALDH2 variants show lower stability and activity in transiently transfected cells. (A) Experimental paradigm; ALDH2 protein levels were determined in transiently transfected 3T3 cells by immunoblotting of total cell lysate. Levels were quantified and presented as ratio vs β−actin (loading control). n = 4; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _** p_-value <0.01; _* p_-value <0.05. (B) Experimental paradigm; ALDH2 protein stability was determined in transiently transfected 3T3 cells by immunoblotting of total cell lysate after 100 μm cycloheximide for 12 h. Levels were quantified and presented as ratio vs β-actin (loading control). n = 4; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _** p_-value <0.01; _* p_-value <0.05. (C)ALDH2 specific activity was determined in transiently transfected 3T3 cells. n = 4; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001 vs other groups.

Fig 4

Fig 4

Novel ALDH2 mutant are sensitive to ethanol toxicity. (A) Cellular ATP levels were measured in transiently transfected 3T3 cells using ATP-based CellTiter-Glo Luminescent Cell Viability kit. n = 8; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _** p_-value <0.01; _* p_-value <0.05. (B) Total cellular ROS levels were measured with 2,7 dichloro- fluorescein diacetate in cells treated as in (A). n = 8; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _** p_-value <0.01; _* p_-value <0.05. (C) Lactate dehydrogenase activity levels in supernatant in cells treated as in (A). n = 8; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _** p_-value <0.01; _* p_-value <0.05. (D) Cellular ATP levels were measured in transiently transfected 3T3 cells in the presence or absence of Alda-1/Alda-64 (20 μM/48 h; 50 mM Ethanol) using ATP-based CellTiter-Glo Luminescent Cell Viability kit. n = 8; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _** p_-value <0.01; _* p_-value <0.05. (E) Total cellular ROS levels were measured with 2,7 dichloro- fluorescein diacetate in 3T3 cells treated as in (D). n = 8; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _** p_-value <0.01; _* p_-value <0.05. (F) Lactate dehydrogenase activity levels in supernatant in 3T3 cells treated as in (D). n = 8; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _** p_-value <0.01; _* p_-value <0.05. (G) 4-HNE protein adducts in transiently transfected human-derived fibroblasts treated as in (D). n = 3; Mean, standard deviation, probability by one‐way ANOVA (with Fischer's LSD post hoc test) _**** p_-value <0.0001; _*** p_-value <0.001; _** p_-value <0.01; _* p_-value <0.05.

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