A simple, rapid, interpretable, actionable and implementable digital PCR based mortality index

Figures & data

Table 1. Key demographic and clinical characteristics of participants

	All	Male	Female
Number of Participants	2256	1022	1234
Age at Intake^†	66.3 ± 8.9 years	66.1 ± 8.8 years	66.5 ± 9.0 years
Current Smoking Status^‡
Yes	179 (8.0)	75 (7.3)	104 (8.4)
No	2074 (91.9)	944 (92.4)	1130 (91.6)
Missing	3 (0.1)	3 (0.3)	0 (0.0)
Past Smoking Status
Yes	203 (9.0)	86 (8.4)	117 (9.5)
No	2050 (90.9)	933 (91.3)	1117 (90.5)
Missing	3 (0.1)	3 (0.3)	0 (0.0)
CHD
Yes	322 (14.3)	201 (19.7)	121 (9.8)
No	1934 (85.7)	821 (80.3)	1113 (90.2)
Diabetes
Yes	271 (12.0)	144 (14.1)	127 (10.3)
No	1978 (87.7)	874 (85.5)	1104 (89.5)
Missing	7 (0.3)	4 (0.4)	3 (0.2)
HbA1c	5.7 ± 0.7%	5.7 ± 0.8%	5.7 ± 0.6%
GrimAge EA	70.1 ± 8.6 years	71.9 ± 8.7 years	68.7 ± 8.3 years
PhenoAge EA	58.8 ± 9.4 years	59.4 ± 9.5 years	58.3 ± 9.4 years
Average Methylation
cg05575921	76.4 ± 8.4%	75.7 ± 9.0%	77.0 ± 7.8%
cg04987734	37.1 ± 5.2%	38.2 ± 5.0%	36.2 ± 5.2%
cg02583484	29.5 ± 3.9%	29.7 ± 4.0%	29.3 ± 3.9%
cg00300879	68.1 ± 7.3%	67.4 ± 7.0%	68.6 ± 7.4%
cg19693031	66.7 ± 5.4%	66.1 ± 5.3%	67.3 ± 5.3%

^†Mean ± Standard Deviation for Continuous Measures; ^‡ N (%) for Categorical Measures

Table 2. Model fit statistics assessing methylation-mortality association

Model	N (Events)	Predictors	Harrell’s C	Pseudo R^2	AIC
1	2274 (289)	Age, Sex	0.744	0.107	4087.3
2	2274 (289)	Model 1 + cg05575921 + cg04987734 + cg02583484	0.783	0.139	4008.0
CHD Marker
3	2274 (289)	Model 2 + cg00300879	0.784	0.141	4005.1
Diabetes Marker
4	2274 (289)	Model 2 + cg19693031	0.786	0.142	4003.3
Multiple Markers: Mortality Index
5	2274 (289)	Model 2 + cg00300879 + cg19693031	0.787	0.144	4000.9
Epigenetic Age & Grim Age
6	2274 (289)	PhenoAge	0.747	0.092	4122.9
7	2274 (289)	Model 1 + PhenoAge	0.757	0.117	4062.9
8	2274 (289)	Grim Age	0.790	0.151	3969.0
9	2274 (289)	Model 1 + Grim Age	0.790	0.151	3972.6

Figure 1. A plot of the relationship of the relationship of the PhenoAge to GrimAge in the FHS cohort (R² = 0.62)

Table 3. Hazard ratio estimates for Model 5

Predictors	HR (95% CI)
Age at Intake^†	2.58 (2.26, 2.95)***
Sex
Male vs. Female	1.38 (1.09, 1.74)**
Average Methylation^†
cg05575921	0.69 (0.62, 0.77)***
cg04987734	1.21 (1.10, 1.32)***
cg02583484	0.89 (0.79, 0.99)*
cg00300879	0.90 (0.81, 0.99)*
cg19693031	0.86 (0.77, 0.97)*

^†Continuous measures are standardized; * p < 0.05, **p < 0.01, ***p < 0.0001

Figure 2. Plots of the relationship of Illumina array methylation values (x-axis) to that of digital PCR values (y-axis) for each of the markers in the predictor set for the subjects in the FACHS study. The R² for each of the correlations with their corresponding Illumina probe values is: Dcg05575921, 0.96; Dcg04987734, 0.93; Dcg19693031, 0.90; Dcg00300879, 0.95

Table 4. Internal validation: bootstrap results

New Marker	Statistic	Original	Training	Test	Optimism	Corrected	Resample
cg00300879	C†	0.7842	0.7872	0.7827	0.0045	0.7797	200
	Shrinkage	1.0000	1.0000	0.9692	0.0308	0.9692	200
cg19693031	C	0.7857	0.7884	0.7839	0.0045	0.7812	200
	Shrinkage	1.0000	1.0000	0.9676	0.0324	0.9676	200

†C = Harrell’s C

Table 5. Demographic characteristics and digital PCR methylation values for the adults subjects from the family and community health study subjects

N	94
Age	48.4 ± 9.5 years
Gender Male Female	25 69
BMI	34.2 ± 8.2
HbA1c	6.1 ± 1.2%
Marker	Average	Dynamic Range
Dcg05575921	74.2 ± 18.6%	29.5 to 91.9%
Dcg19693031	76.9 ± 5.8%	58.1 to 87.9%
Dcg04987734	36.1 ± 7.4%	20.7 to 57.3%
Dcg02583484	23.7 ± 5.3%	13.1 to 38.4%
Dcg00300879	67.1 ± 14.1%	3.2 to 90.9%

Table 6. Demographic and digital PCR methylation values for adolescents from the health iowa study

	African Americans	White	Nominal p-value	Corrected p-value
N	45	45
Age	15.8 ± 0.4 years	15.6 ± 0.6 years	P < 0.55	-
Gender Male Female	24 21	21 24
BMI	24.4 ± 4.7	22.3 ± 3.2	P < 0.0001	-
Dcg05575921	86.0 ± 2.9%	86.4 ± 2.8%	P < 0.55	N.S.
Dcg19693031	74.8 ± 4.2%	75.3 ± 3.8%	P < 0.51	N.S.
Dcg04987734	29.3 ± 5.4%	28.3 ± 6.7%	P < 0.46	N.S.
Dcg02583484	27.5 ± 5.1%	25.2 ± 4.7%	P < 0.04	N.S.
Dcg00300879	61.3 ± 11.4%	56.6 ± 8.9%	P < 0.04	N.S.