CLINICAL RESEARCH e-ISSN 1643-3750 © Med Sci Monit, 2016; 22: 316-324 DOI: 10.12659/MSM.894912

VEGF Gene Polymorphisms Affect Serum Protein Levels and Alter Disease Activity and Synovial Lesions in Rheumatoid Arthritis

Received: 2015.06.05 Accepted: 2015.08.12 Published: 2016.01.30

Authors’ Contribution: Study Design  A Data Collection  B Analysis  C Statistical Data Interpretation  D Manuscript Preparation  E Literature Search  F Collection  G Funds

ACEG 1,2 ABD 1,3 BDF 1 BDF 1 BDG 1,2 ADF 1



Corresponding Author: Source of support:



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Jin-Ping Yi Yu-Zhang Wu Nan Yu Zhi-Wu Yu Fu-Yuan Xie Quan Yuan

1 Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, P.R. China 2 Division of Laboratory Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, P.R. China 3 Institute of Immunology, College of Basic Medical Sciences, The Third Military Medical University, Chongqing, P.R. China

Yu-Zhang Wu, e-mail: [email protected] This study was funded by National Key Basic Research Program (No. 2010CB529102)

Our study investigated 2 common single-nucleotide polymorphisms (SNPs) of vascular endothelial growth factor (VEGF) for their influences on serum VEGF levels, disease activity, and synovial lesions in rheumatoid arthritis (RA). Clinical information and venous blood samples were collected from 98 RA patients and 100 healthy controls. Genotyping on samples from the subjects was performed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Serum VEGF levels were determined using the enzyme-linked immunosorbent assay (ELISA). The synovial thickness and joint effusion of 28 joints were measured in RA patients, and total sharp score (TSS) and disease activity score (DAS) of 28 joints were recorded. The genotype and allele frequencies of VEGF rs833070 (G>A) and rs3025030 (G>C) were significantly different between RA group and control group (all PC)] for their influences on the circulating levels of VEGF protein and their effects on disease activity and synovial lesions in RA.

Material and Methods Ethics statement The study was approved by the Ethics Committee of the Zhujiang Hospital, Southern Medical University. The written informed consent was provided by each eligible patient and the study conformed to the Declaration of Helsinki [12].

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License

Clinical data collection General clinical data of all the study subjects such as age, sex, disease duration, present and past medical history, and the history of hereditary disease were collected and recorded. Common clinical laboratory parameters for RA including routine blood test, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) and acute C-reactive protein (CRP) were collected. Detection of VEGF polymorphisms Morning fasting venous blood samples (2 ml) were collected from all subjects. EDTA was used as an anticoagulant for blood collection. Genomic DNA was extracted from the white blood cells (WBCs) collected from venous blood by using a DNA Extraction Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The SNP genotyping was conducted using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (Sequenom Inc., San Diego, CA, USA) [14]. The primers (Table 1) were designed with MassARRAY® Assay design 3.1 software, available with the MassARRAY SNP genotyping system (Sequenom, Inc., San Diego, CA, USA). The PCR reaction condition was: 45 cycles of predenaturation at 94°C for 15 s, denaturation at 94°C for 20 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 3 min, and stored at 4°C. SAP reaction solution was prepared and added into a 384-well plate containing PCR-amplified products to degrade dNTP at 37°C for 20 min, then placed at 85°C for 5 min

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Yi J.-P. et al.: VEGF & disease activity and synovial lesions in RA © Med Sci Monit, 2016; 22: 316-324

CLINICAL RESEARCH

Table 1. Primer sequence and primer length (bp) of rs833070 (G/A) and rs3025030 (G/C) in the vascular endothelial growth factor gene. SNP

Primers

Primer length (bp)

5’-ACGTTGGATGAAGTTCACAGCACCCGAACA-3’ rs833070 (G/A)

86 5’-ACGTTGGATGCCCTGGTTTGCATTCCTTTG-3’ 5’-ACGTTGGATGAAAATGTGTGGGCTGCTTGG-3’

rs3025030 (G/C)

111 5’-ACGTTGGATGACACACTGAAGGAGCTGTAG-3’

to inactivate SAP, and stored at 4°C. Next, the iPlex reaction system was prepared and added into the 384-well plate, and the single-base extension reaction was conducted under the following conditions: at 94°C for 30 s, at 94°C for 5 s, at 52°C for 5 s, at 80°C for 5 s, at 72°C for 3 min, and storing at 4°C. The purification of PCR products was performed by demineralization with resin. The DNA samples were transferred from the 384-well plate onto the MassARRAY SpectroCHIP covered with matrix, and detected with the mass spectrometer. The genotyping analysis was performed by using the MassARRAY® TYPER software.

An experienced physician with ultrasound expertise, who did not know the disease status and treatment status of the patients, monitored the ultrasound examinations. Ultrasonic examination

The serum concentrations of VEGF were measured by the ELISA method using an ELISA kit provided by Wuhan Boshide Company (Wuhan, China) in accordance with the manufacturer’s instructions [15].

Based on the classification standard of Walther, the synovial thickness was classified into 4 grades: grade I, no synovial hyperplasia and thickness 9 mm [17]. Joint effusion in joint scotoma (anteroposterior diameter) was determined based on different joint size and position: knee suprapatellar bursa scotoma >4 cm is considered as joint effusion; medial and lateral knee scotoma >2 cm as joint effusion; scotoma of shoulder joins, elbow joints, and wrist joints, metacarpophalangeal and proximal interphalangeal joints >2 cm as joint effusion.

Disease activity score (DAS)

Power Doppler evaluation

A total of 28 joints, including shoulders, elbows, wrists, metacarpophalangeal joints, proximal interphalangeal joints of hands, and knee joints, were scored by DAS scoring system [16]. The number of swollen and tender joints in each RA patient was examined and recorded, and the DAS28 score was calculated by combining the ESR and self-evaluation in patients with RA. [n1, the number of tender joints; n2, the number of swollen joints; n3, ESR (mm/h); n4, health evaluation in RA patients]. The DAS28 score of 5.1 as frequent mobility.

The semi-quantitative signal of each point of synovium in the 28 joints was observed and classified: grade 0, no signal or blood flow; grade 1, slight signal, and single or independent vascular signals less than 3; grade 2, moderate fusion vessel, and more than 3 independent signals, or fusion signals less than half the synovial region; grade 3, significant signal, and visible vascular signals in more than half the medial region of the joints.

Enzyme-linked immunosorbent assay (ELISA)

Ultrasonography The ultrasonic examination was performed using a Siemens ACUSON S2000™ color Doppler (Siemens, Erlangen, Germany), with the probe frequency of 8–13 MHZ. The 28 joints of each RA patient were examined using direct contact method. All patients adopted the supine or sitting position, adjusted based on the examined joints, with the examined joint fully exposed.

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Recording items Recording items consisted of the followings: (1) US joint count SH: the number of joints with thickened synovium; (2) US joint count SF: the number of joints with effusion; (3) US joint count PD: the number of joints with energy signals; (4) US index SH: total synovial thickening score, which was the sum of the synovial thickening score of the 28 joints; (5) US index SF: total joint effusion score, which was the sum of joint effusion score of the 28 joints; (6) total sharp scores (TSS): the sum of the highest power Doppler score of each joint.

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Yi J.-P. et al.: VEGF & disease activity and synovial lesions in RA © Med Sci Monit, 2016; 22: 316-324

CLINICAL RESEARCH

Table 2. Comparison between demographic and clinical characteristics of patients with rheumatoid arthritis and demographic features of healthy controls. Variable Age (years)

Control group (n=100)

RA group (n=98)

48.67±13.41

Gender (male/female)

50.82±12.53

35/65

32/66

P 0.245 0.727

WBC (×109/L)

6.31±1.88

6.81±2.88

0.114

RBC (×10/L)

4.69±0.31

4.64±0.66

0.498

PLT (×10/L)

276.6±64.5

282.55±65.9

0.521

HGB (g/L)

129.6±12.0

127.0±11.9

0.125

ESR (mm/h)

16.31±2.75

36.39±3.44

0.05). The distributions of allele and genotype frequencies of the VEGF rs833070 (G>A) in the RA group were significantly different from the control group (all P

VEGF Gene Polymorphisms Affect Serum Protein Levels and Alter Disease Activity and Synovial Lesions in Rheumatoid Arthritis.

Our study investigated 2 common single-nucleotide polymorphisms (SNPs) of vascular endothelial growth factor (VEGF) for their influences on serum VEGF...
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