Elsevier

Bone

Volume 71, February 2015, Pages 36-41
Bone

Original Full Length Article
Genome-wide pathway-based association study implicates complement system in the development of Kashin-Beck disease in Han Chinese

https://doi.org/10.1016/j.bone.2014.09.025Get rights and content

Highlights

  • CACC pathway is significantly associated with KBD.

  • rs1656966 in CACC pathway is significantly associated with KBD in 1026 subjects.

  • CFD, A2M, C5 and CD46 genes of CACC pathway are up-regulated in KBD cartilage.

  • Serum C5 in KBD patients is significantly higher than that in healthy controls.

  • CACC pathway contributes to the development of KBD.

Abstract

Kashin-Beck disease (KBD) is a chronic osteochondropathy. The pathogenesis of KBD remains unknown. To identify relevant biological pathways for KBD, we conducted a genome-wide pathway-based association study (GWPAS) following by replication analysis, totally using 2743 Chinese Han adults. A modified gene set enrichment algorithm was used to detect association between KBD and 963 biological pathways. Cartilage gene expression analysis and serum complement measurement were performed to evaluate the functional relevance of identified pathway with KBD. We found that the Complement and Coagulation Cascades (CACC) pathway was significantly associated with KBD (P value = 3.09 × 10 5, false-discovery rate = 0.042). Within the CACC pathway, the most significant association was observed at rs1656966 (P value = 1.97 × 10 4) of KNG1 gene. Further replication study observed that rs1656966 (P value = 0.037) was significantly associated with KBD in an independent validation sample of 1026 subjects. Gene expression analysis observed that CFD (ratio = 3.39 ± 2.68), A2M (ratio = 3.67 ± 5.63), C5 (ratio = 2.65 ± 2.52) and CD46 (ratio = 2.29 ± 137) genes of the CACC pathway were up-regulated in KBD articular cartilage compared to healthy articular cartilage. The serum level of complement C5 in KBD patients were significantly higher than that in healthy controls (P value = 0.038). Our study is the first to suggest that complement system-related CACC pathway contributed to the development of KBD.

Introduction

Kashin-Beck disease (KBD) is a chronic osteochondropathy [1], [2], which first occurs in children (aged 3–12 years). KBD patients suffer from serious joint pain, joint deformities, osteoarthritis and growth retardation [3], [4]. Currently, KBD is mainly prevalent in some areas of China, Russia and North Korea. In China, there are more than 2.5 million KBD patients, and about 30 million people living in KBD prevalent areas are at the risk of KBD [5]. According to the extent of joint lesions, KBD is clinically classified into grade I, II and III KBD [5]. Because the pathogenesis of KBD remains unknown, there is not curative approach for KBD now.

Recent studies found that genetic factors played an important role in the development of KBD. Lv et al. observed significantly familial clustering of KBD patients in a large cohort of 10,823 subjects from 1361 Han Chinese families [4]. The estimated heritability of KBD varied from 35.10% to 41.76% in families [4], [6]. Candidate gene association studies reported several susceptibility genes for KBD [7], [8]. However, the genetic basis of KBD is largely unknown. Genome-wide interrogating the genetic variants affecting the risk of KBD can help to clarify the pathogenesis of KBD. Recently, genome-wide pathway-based association studies (GWPAS) were proposed [9], and successfully applied in the genetic studies of complex diseases [10], [11], [12], [13], [14]. Identifying disease-relevant biological pathways can provide novel insight into the pathogenesis of complex diseases [15]. To the best of our knowledge, no GWPAS of KBD has been reported by now, limiting our efforts to clarify the pathogenesis of KBD.

In this study, a GWPAS of KBD was first conducted in 1717 Chinese Han subjects. The most significant association signal in KBD-associated pathway was then validated in an independent validation sample containing 1026 subjects. Gene expression analysis of KBD articular cartilage and serum complement measurement were also performed to evaluate the functional relevance of identified pathway with KBD. We found that the Complement and Coagulation Cascades (CACC) pathway was significantly associated with KBD. To the best of our knowledge, this study is the first to suggest that the CACC pathway contributed to the development of KBD.

Section snippets

Human subjects

Extreme phenotype sampling was used in the GWPAS [16]. Fifty-nine grade II and 31 grade III unrelated KBD patients with extreme KBD phenotypes were recruited from Linyou county of Xi'an city of Shaanxi province in China. All of the 90 KBD patients presented similar extreme KBD phenotypes, including shortened phalanges, shortened humeri and serious short stature (Fig. 1). A total of 1627 unrelated healthy subjects recruited from Xi'an city and Changsha city in China, were used as control samples

Results

STRUCTURE analysis observed that all GWPAS samples were clustered together as a homogeneous population. EIGENSTRAT analysis obtained a genomic control inflation factorλgc = 1.03, indicating no significant population stratification in our GWPAS samples.

Among the 963 pathways analyzed in this study, the CACC pathway showed the most significant association with KBD (P value = 3.09 × 10 5) (Fig. S1). After adjusting for multiple testing, the CACC pathway was still significantly associated with KBD (FDR

Discussion

In this study, we conducted a GWPAS of KBD and found that the CACC pathway was significantly associated with KBD. Further replication study validated the most significant association signal observed in the CACC pathway. Gene expression analysis also found that A2M, CFD, C5 and CD46 genes of the CACC pathway were up-regulated in KBD articular cartilage compared to healthy articular cartilage. The serum C5 level of KBD patients appeared to increase compared to healthy controls. Our results

Acknowledgments

The study was supported by National Natural Scientific Fund of China (81102086), Science and Technology Research and Development Program of in Shaanxi Province of China (2013KJXX-51), Natural Science Basic Research Program Shaanxi Province (2011JQ4013).

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