may not be a predictor of risk of ischemic vascular APOC 3 disease in the Chinese population

The genetic background of ischemic vascular disease is actively being explored. Several studies have shown that inhibition of significantly APOC3 reduces plasma levels of apolipoprotein C3 and triglycerides. Recently, the TG and HDL Working Group and Jørgensen reported that loss-of-function et al. mutations in are associated with decreased triglyceride levels and a APOC3 reduced risk of ischemic vascular disease in European and African individuals. We performed a replication study in 4470 Chinese participants. The coding regions of were amplified and re-sequenced. However, only APOC3 synonymous and intronic variants with no functional consequences were identified. None of the loss-of-function mutations reported in European and African individuals were observed. Therefore, may not be an ideal APOC3 predictor for risk of ischemic vascular disease in the Chinese population. 1,2* 1,2* 1 1,2 1 1 1,2

The genetic basis of ischemic vascular disease such as coronary artery disease is actively being explored. Most studies have focused on susceptibility factors contributing to an increased risk 1 , while only a few studies have identified protective variants conferring reduced risk 2 . Recently, the TG and HDL Working Group and Jørgensen et al. reported that loss-of-function mutations in APOC3 are associated with decreased triglyceride levels and a reduced risk of ischemic vascular disease in individuals of European and African ancestry 3,4 . Approximately 1 in 150 individuals were heterozygous carriers of at least one of the four mutations: R19X, A43T, IVS2+1G→A, and IVS3+1G→T. Heterozygous carriers for these mutations had a significantly lower incidence of ischemic vascular disease as compared to non-carriers (hazard ratio = 0.59). Triglyceride and circulating APOC3 levels in the carriers were only 61% and 54% of those in non-carriers, respectively. These critical findings prompted us to undertake a replication study in China, where over one million people are affected by cardiovascular diseases each year (http://www.nhfpc.gov.cn/).
A total of 4470 unrelated Chinese participants were enrolled, including 1488 healthy controls, 1050 patients with ischemic stroke, 628 patients with coronary artery disease, and 1304 patients with venous thrombosis, which could also be exacerbated by effects on the coagulation system resulting from elevated triglyceride levels.
The 1488 healthy controls and 1050 patients with ischemic stroke were described in a previous study 5 . Briefly, healthy individuals did not present any relevant medical history or family history of ischemic vascular disease. Ischemic stroke was confirmed by brain computed tomography (CT) and/or brain magnetic resonance imaging (MRI). The 1304 patients with venous thrombosis were described previously 6 . Thrombosis was confirmed by objective investigations such as color Doppler ultrasonography and/or CT angiography. Patients with coronary artery disease were enrolled in our hospital from September 2013 to March 2014. Coronary artery disease was validated by angiographic evidence of at least one segment of a major coronary with over 50% organic stenosis. The characteristics of the 628 patients with coronary artery disease are summarized in Table 1. Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of Union Hospital affiliated with Huazhong University of Science and Technology (Approval number 2013-03-0052).
Blood samples were collected into a vacutainer tube containing 0.105 mol/L trisodium citrate and were then centrifuged at 2000 g for 15 minutes. Genomic DNA was isolated using a salt precipitation method and was then used for sequencing. The four exons and the flanking intronic regions of APOC3 were amplified by PCR and then sequenced on an Applied Biosystems ABI 3730 Genetic Analyzer, as previously described 3 . The oligonucleotide pairs and annealing temperatures employed in PCR and sequencing are shown in Table 2. In this study, we identified only synonymous and intronic variants, with no functional consequences, and similar genotype distributions across the groups ( Table 3). None of the loss-of-function mutations reported in European and African individuals were observed in the current cohort. Therefore, the genetic background of ischemic vascular disease is highly variable among different ethnic groups, and APOC3 may not be an

REVISED Amendments from Version 1
In response to the reviewers' comments, we have added some important limitations to the study in the revised text. The diagnosis of myocardial infarction was based on typical chest pain with a duration over 30 min, on electrocardiographic patterns, and on increased creatine kinase MB isoenzyme and troponin I levels. Hypertension is defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. Type 2 diabetes were clarified using the 1999 WHO criteria, including fasting plasma glucose ≥ 7.0 mmol/L, 2-hour oral glucose tolerance test plasma glucose ≥ 11.1 mmol/L or ongoing therapy for diabetes.  ideal predictor of risk of ischemic vascular disease in the Chinese population.

See referee reports
However, there are some limitations in this study. First, we only analyzed the coding sequence, as in the studies carried out in European and African individuals. Potential effects from large deletions and mutations in regulatory regions of the gene cannot be excluded. Previous studies have revealed two promoter polymorphisms (T-455C and C-482T) that affect the expression of APOC3. However, their relationship with coronary artery disease was not observed in a Han Chinese population 7 . Second, the consequences of the synonymous mutations and the intronic mutations identified here were only predicted by an on-line bioinformatics tool (http://www.fruitfly.org/seq_tools/splice.html). Third, the triglyceride levels were not available for the patients with venous thrombosis. Thus, the triglyceride levels according to the genotypes were not further analyzed. Nevertheless, considering the relatively large sample size, we suggest that functional variants in APOC3 could be very rare in China. Further studies are warranted to understand the genetic basis governing triglyceride levels and conferring protective effects on ischemic vascular disease in the Chinese population.

Consent
Written informed consent to publish these data has been obtained by each participant.
Author contributions YH and LT chose the article for correspondence and evaluated the data in the manuscript. LT, ZPC, QYW, WZ, HL, YYW, and BH performed experiments. LT and ZPC wrote the manuscript. YH supervised the process and critically edited the manuscript.

Competing interests
No competing interests were disclosed.

Grant information
This study was supported by grants from the National Natural Sciences Foundation of China (No. 81370622 and No. 81400099) and the Independent Innovation Foundation of Huazhong University of Science and Technology (No. 01-18-530045, 2013QN213).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
should clearly state reasons why they believe that the variants identified in the study have no functional consequences.
References should be added to the end of the second sentence of the paper (while only "a" few studies have identified protective variants conferring reduced risk).
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed. Two very recent landmark large-scale studies show that loss-of-function mutations in APOC3 are associated with lower levels of plasma triglycerides, and carriers of these mutations have a reduced risk of coronary heart disease in European and African individuals. Tang . performed a timely replication et al study in 4,470 Chinese individuals. Unexpectedly, no loss-of-function mutations identified in the European and African populations were found in the Chinese population. This important study not only highlights the difference in genetic susceptibility to cardiovascular disease in different ethnic populations, but also suggests that APOC3 variants are not applicable to the Chinese population to predict risk for ischemic vascular disease.
APOC3 may still be an important regulator of lipid metabolism in Chinese, and novel variants of this gene remain to be identified in this ethnic population. Consequently, the authors need to change their conclusion from "Therefore, may not be an ideal predictor for risk of ischemic vascular disease in APOC3 the Chinese population" to "Therefore, variants identified in the European and African population APOC3 may not be an ideal predictor for risk of ischemic vascular disease in the Chinese population".

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. Competing Interests: