ACE-Dependent Alzheimer’s Disease: Blood ACE Phenotyping of the Most Prevalent and Damaging ACE Missense Mutation—Y215C (rs3730025)

Figures

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  Figure 1. Quantification of blood ACE levels in carriers of controls and the Y215C ACE mutation. Blood ACE protein was precipitated from EDTA plasma by mAb 9B9, and its activity was then quantified fluorometrically using ZPHL as a substrate. (A) The immunoreactive ACE protein was quantified in plasma samples from 48 controls. (B) The immunoreactive ACE protein—from 84 carriers of the Y215C ACE mutation corrected for genotype [16,19]. ACE levels were presented as the means ± SD of several independent assessments of samples. The data are expressed as % of ACE levels of the corresponding value for control pooled plasma samples from donors without ACE mutations. Orange, brown, and red bars indicate samples with ACE levels > 120%, 150%, and 200% of controls, respectively. Yellow and blue bars—samples with ACE levels < 80% and 50% of controls, respectively. Gray bars—ACE levels between 80% and 120% of the control values. The dashed line marks the 100% control baseline.

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  Figure 2. ACE precipitation by mAbs from the EDTA plasma pools obtained from Y215C ACE mutation carriers. Blood ACE protein was precipitated using 3 mAbs targeting the N domain (9B9, 1G12, and 5B3). Plasma pools were combined according to the data shown in Figure 1A. Samples with ACE levels less than 80% and less than 50% of the control were combined into the blue and yellow pools, respectively. Pools with ACE levels near 100% were combined into the gray pool, and samples with levels greater than 120% and greater than 200% of the control were combined into the orange and red pools, respectively. (A) Precipitated ACE activity by mAb 9B9, and its activity, was then quantified fluorometrically using ZPHL as a substrate. (B) Precipitated ACE activity by mAb 1G12, the same substrate. (C) Precipitated ACE activity by mAb 5B3, the same substrate. (D) 5B3/1G12 binding ratio. Coloring is as described in the legend to Figure 1. The dashed line marks the 100% control baseline.

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  Figure 3. Quantification of blood ACE levels in carriers of the ADAM9 mutations. (A) The immunoreactive ACE protein was performed in plasma samples from 11 carriers of 10 different mutations in ADAM9. (B) Plasma ACE levels adjusted according to the donor’s genotype for the I/D polymorphism ACE levels were presented as the means ± SD of several independent assessments of samples. The data are expressed as % of ACE levels of the corresponding value for control pooled plasma samples from donors without ACE mutations. Bottom: Predictions of the potential damaging effects of mutations on the ACE protein were derived from Table S1 [16,19] and based on Poly-Phen2, AlphaMissense, and CADD. For AlphaMissense and CADD: pathogenic predictions are shown in red, uncertain significance in yellow, and benign predictions in green. For Poly-Phen-2 specifically: purple is used for both the ‘possibly damaging’ (score 0.446–0.908) and ‘benign’ (score ≤ 0.445) categories, while ‘probably damaging’ (score ≥ 0.909) is shown in red. Coloring is as described in the legend to Figure 1. The dashed line marks the 100% control baseline.

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  Figure 4. Quantification of blood ACE levels in carriers of the ADAMTS13 mutations. (A) The immunoreactive ACE protein was quantified in plasma samples from 11 carriers of 7 different mutation in ADAMTS13. (B) Plasma ACE levels adjusted according to the donor’s genotype for the I/D polymorphism ACE levels were presented as the means ± SD of several independent assessments of samples. The data are expressed as % of ACE levels of the corresponding value for control pooled plasma samples from donors without ACE mutations. Bottom: Predictions of the potential damaging effects of mutations on the ACE protein were derived from Table S1 [16,19] and based on AlphaMissense and CADD. Pathogenic predictions are shown in red, uncertain significance in yellow, and benign predictions in green. Coloring is as described in the legend to Figure 1. The dashed line marks the 100% control baseline.

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  Figure 5. Quantification of blood ACE levels in carriers of the MMP21 mutations. (A) The immunoreactive ACE protein was quantified in plasma samples from 6 carriers of 5 different mutations in MMP21. (B) Plasma ACE levels adjusted according to the donor’s genotype for the I/D polymorphism ACE levels were presented as the means ± SD of several independent assessments of samples. The data are expressed as % of ACE levels of the corresponding value for control pooled plasma samples from donors without ACE mutations. Coloring is as described in the legend to Figure 1. The dashed line marks the 100% control baseline.