Haptoglobin genotype predicts development of coronary artery calcification in a prospective cohort of patients with type 1 diabetes

The Coronary Artery Calcification in Type 1 Diabetes (CACTI) study enrolled 1,416 individuals between 19 and 56 years of age, with no known history of CAD: 652 participants with type 1 diabetes and 764 control participants without diabetes. Twenty five people were excluded because they had a coronary event. Of those remaining, 172 individuals with type 1 diabetes and 216 controls have not completed the 6 year follow-up examination. Additional subjects were excluded because of missing plasma sample (n = 28) or inability to determine the Hp genotype (n = 13), leaving 436 subjects with diabetes and 526 controls in the analyses. The baseline CAD risk factors for the CACTI participants included in the analyses did not differ from those who were excluded, except for younger age of excluded controls. Participants with type 1 diabetes had long-standing disease (mean duration ± SD: 23 years ± 9 years) at baseline, were insulin dependent within 1 year of diagnosis, and were diagnosed prior to age 30 years or had positive antibodies or a clinical course consistent with type 1 diabetes. All study participants provided informed consent and the study protocol was approved by the Colorado Multiple Institutional Review Board.

At each examination, an ultrafast Imatron C-150XLP EBCT scanner (Imatron, San Francisco, CA) was used to obtain two sets of high resolution, noncontrast, contiguous 3-mm tomographic images acquired at 100-ms exposure. Scanning started from near the lower margin of bifurcation of the main pulmonary artery with the subject breathholding for ~35-45 s and proceeded caudally. Calcified coronary artery areas were identified as those with a minimum density of 130 Hounsfeld units (HU) and a minimum area of three pixels (1.03 mm2). A calcium score for each region was calculated by multiplying the area by the density score (1 for 130-199, 2 for 200-299, 3 for 300-399, and 4 for > 399 HU). A total CAC score in Agatston units (AU) was calculated by adding up scores for all slices separately for left main, left anterior descending, circumflex, and right coronary arteries [14]. The scanner was recalibrated every day with a phantom. Effective radiation dose for an EBCT sequence was 1.0 mSV for men and 1.3 mSV for women [15].

The presence of any CAC at baseline was defined as a CAC score > 0 on either of the two scans. CAC progression was defined as a change in square root CAC volume ≥ 2.5, as this difference was determined to be < 1% likely to be due to measurement error, based on the two scans that were completed within 5 minutes of one another [16]. The same CAC measurement protocol was followed at the baseline and 6 year follow up visit.

Hp typing was performed on stored plasma samples by polyacrylamide gel electrophoresis as previously described [17]. Briefly, 10 ul of Hb enriched plasma was subjected to electrophoresis in a non-denaturing gel and the gel was subsequently immersed in solution containing a congener of benzidine with a precipitate forming in the gel corresponding to the location of Hb-Hp complexes. The Hp type of the sample was determined by the banding pattern of the Hp-Hb complexes with each of the three Hp types having a characteristic banding fingerprint. Previous work has established a 1:1 correspondence between this method and a PCR based method for Hp genotyping [17]. An unambiguous Hp type was obtained on 98.7% of all samples. For the purposes of quality and control and validation, we simultaneously measured the Hp type using an ELISA based assay [7, 18] with a greater than 97% agreement in the Hp type assigned by the two methods.

We used two analytic approaches to examine the association between CAC and Hp polymorphism: linear regression and logistic regression. For both analyses, we considered the following baseline CAD risk factors as covariates: sex, age, diastolic blood pressure (mmHg), systolic blood pressure (mmHg), body mass index (Kg/m²) (BMI), low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides, hemoglobin A1c (%), self-reported history of ever smoking, and baseline square root transform of the CAC volume among those with CAC present at the baseline examination. We used backwards selection to build the most parsimonious model; this selection process entails removing the covariate that has the largest p-value given the other covariates in the model until all covariates included have a p value < 0.05. Any covariate whose p-value was not < 0.05 in the multivariate model was not included in the final adjusted model. Assessment of baseline CAD risk factors have been previously described [15]. The outcome in linear regression analyses was the change in square root transformed CAC volume over the 6 year follow up period. For logistic regression analyses, CAC progression was defined as a change in the square root transformed CAC volume ≥ 2.5 over the 6 year period of follow-up, which corresponds to a change in CAC volume from 0 to ≥ 6.25 among participants initially free from CAC. This cut off was chosen because it is the point at which the change in CAC is significant enough that it can not be attributed to interscan variability [16]. All analyses were performed using SAS for Windows version 9.2 (SAS Institute Inc., Cary, NC, USA).

The Hp 2-2 polymorphism predicted change in CAC volume only in subjects with type 1 diabetes who were free of CAC at the baseline visit (Table 2, bold type). The findings were similar with adjustment for age and sex only (p = 0.03) and when additionally adjusting for systolic blood pressure and HDL-cholesterol (p = 0.05). Hp polymorphism was not associated with change in CAC volume in subjects with type 1 diabetes with CAC present at baseline or in subjects without diabetes, regardless of CAC extent at baseline.

Similar to the above findings, the Hp 2-2 polymorphism predicted development of CAC (a change in CAC volume from 0 to ≥ 6.25 or in square root transformed CAC volume from 0 to ≥ 2.5) only in subjects with type 1 diabetes free of CAC at the baseline examination when adjusting for age and sex (OR: 2.03, 95% CI: 1.13-3.65, p = 0.02), and when additionally adjusting for systolic blood pressure and HDL cholesterol (OR: 1.95, 95% CI: 1.07-3.56, p = 0.03) compared to those without the Hp 2-2 polymorphism (Table 3). Hp polymorphism did not predict CAC progression in individuals without diabetes.

Among patients with diabetes free of CAC at baseline, Hp 2 appeared to have an allele-dose effect on development of CAC: Hp 2-1 OR: 1.72 (1.09-2.71) and Hp 2-2 OR: 2.94 (1.87-4.65), compared to Hp 1-1 and adjusting for age and sex (Figure 1).