Possible association of single nucleotide polymorphisms in the 3' untranslated region of HOXB9 with acetabular overcoverage

Article focus

To date, an association between acetabular over-coverage and genetic variations has not been studied. We investigated the association between single nucleotide polymorphisms (SNPs) of paralogous HOX9 genes and acetabular over-coverage.

Key messages

The HOXB9 SNPs might be involved in the morphogenesis of acetabular coverage, and could be an independent risk factor for developing pincer-type femoroacetabular impingement.

Strengths and limitations

Strengths: This is the first report on the association of genetic variations in acetabular over-coverage. The HOXB9 SNPs showed significant association with acetabular over-coverage compared with controls.

Limitations: More studies should be conducted with larger sample numbers and using different ethnic groups.

Introduction

The hip is a ball-and-socket joint. The socket is formed by the acetabulum, which is part of the large bone of the pelvis. The degree of acetabular coverage is correlated with several hip diseases, such as acetabular dysplasia (AD), femoroacetabular impingement (FAI), labral tears, cartilage damage, and hip osteoarthritis (OA).^1-3 AD is a major and common cause of secondary hip OA, particularly in women.¹ It is characterised by a shallow acetabulum and decreased coverage of the femoral head. This condition appears to be related to a number of different factors.² Several studies have investigated the association between genetic variations and AD.^4-10 On the other hand, the morphogenesis of acetabular over-coverage (AO) remains poorly understood. Excessive acetabular coverage has been reported to be related to FAI (pincer morphological features).^3,11,12

FAI is characterised by contact arising from abnormal morphological features of the hip joint, and has been associated with early OA of the hip. Patients with FAI are young, usually in their 20s to 40s. The estimated prevalence of FAI is 10% to 15%.¹³ The morphological mechanism of FAI is well established.³ However, the genetic predisposition in the pathogenesis of FAI remains unknown. Two main types of FAI have been described in relation to morphological variations of the acetabulum and femoral head, namely: pincer-type and cam-type impingement. Pincer-type impingement occurs on the excessive acetabular coverage, and is more common in women.¹¹ AO is correlated with the radiologic depth of the acetabular fossa. To date, an association between AO and genetic variations has not been studied. Considering the involvement of genetic variations in several examples of AD studies, it is worth investigating whether any genetic variation is associated with susceptibility to AO.

Homeobox (HOX) genes encode a family of transcription factors of fundamental importance for body patterning along the anteroposterior (AP) axis during skeletal development.^14,15 Particularly, paralogous Hox9 genes were shown to have an essential role in the development of the limb buds.^16-19 In the present study, to identify a possible genetic variation associated with AO, we investigated the association between single nucleotide polymorphisms (SNPs) of paralogous HOX9 genesand AO.

Patients and Methods

Results

In 316 subjects, 130 were AO-positive cases and 186 were controls. As shown in Table I, 32.3% of AO cases had acetabular retroversion (AR OR = 6.92, p = 1.87E-9, 95% CI 3.47 to 13.8). We performed genotyping of 19 SNPs in the four HOX9 paralogs (Table II) by PCR amplification using forward and reverse primers. We found a novel SNP located in the 3’ UTR of HOXB9, c1486G> A (rs79931349). In this study, we focused on seven specific SNPs located in the 3’ UTR of HOXB9 that were significantly associated with AO (Tables III and IV). The minor allele frequencies for these SNPs were all > 0.05. The statistical power was 80% at the effect size 0.158.^25-28 The remaining 12 SNPs in the HOX9 paralogs did not show a significant association with AO (data not shown).

In the single-locus association analysis, distributions of genotypes in AO cases and controls were all in accordance with the Hardy–Weinberg equilibrium (Table III). The genotype and allele frequencies of the five HOXB9 SNPs (rs8844, rs3826541, rs3826540, rs7405887, rs79931349) showed statistically significant differences between AO cases and controls (rs7405887 OR = 3.16, p = 5.29E-6, 95% CI 1.91 to 5.25). On the other hand, the two SNPs (rs2303485, rs2303486) were associated with the decreased acetabular coverage (rs2303485 OR = 0.524, p = 0.0091, 95% CI 0.322 to 0.855; rs2303486 OR = 0.519, p = 0.011, 95% CI 0.312 to 0.865) (Table IV). Subsequently, we carried out a haplotype-based association study using Haploview,²¹ which was used to visualise the structure of pair-wise LD between the SNPs, and to define haplotype blocks. A total of five SNP pairs showed high LD values, and constituted a single haplotype block in the 3’ UTR of HOXB9 (Fig. 1). Results of the haplotype analysis for HOXB9 are presented in Table IV. A total of seven possible haplotypes were obtained, five of which had a frequency < 5%; therefore, we analysed the two most prevalent haplotypes (C-C-T-C-G/A-A/T-G and T-A-C-G-G/A-A/T-A). The haplotype with minor alleles of five SNPs (T-A-C-G-G/A-A/T-A) showed a significant association with AO (OR = 2.59, p = 2.61E-5, 95% CI 1.65 to 4.08).

Fig. 1

LD plot of seven single nucleotide polymorphisms (SNPs) in the 3’ UTR of HOXB9. r²values that correspond to SNP pairs are expressed as percentages and shown within the respective squares. Higher r² values are indicated in red. These five SNPs constitute a haplotype block that spans the 3’ UTR of the HOXB9 gene.

Discussion

Excessive acetabular coverage is thought to be the major cause of pincer-type FAI.^3,11,12 While AD has been reported to be associated with several genetic variants,^4-10 the gene variants of AO remain unclear. Nevertheless, prior to our study, no other studies have investigated the association between genetic variations and AO. To the best of our knowledge, this is the first report on the association of genetic variations of HOX9 in AO.

The mammalian Hox complex contains 39 genes organised into four different clusters, Hox A, B, C, and D. Based on DNA sequence and the position of the genes on their respective chromosomes, individual members of the four clusters have been classified into 13 paralogous families. Members of a paralogous family often share similar gene expression patterns. The chromosomal order of the HOX genes is collinear to the relative position of their expression domains along the AP axis of the developing embryo, such that the more 3’ the gene is located, the more anterior its expression boundary.^14,15,29 Using gene targeting, abdominal B-related genes, which are paralogous to Hox9-13 genes, were shown to have an essential role in the development of the limb buds.^16,30,31 In humans, Muragaki et al³² demonstrated that synpolydactyly, an inherited abnormality of the hands and feet, is caused by expansion of a polyalanine stretch in the amino–terminal region of HOXD13. Mortlock and Innis³³ reported the identification of a HOXA13 nonsense mutation in a family with hand–foot–genital syndrome. Paralogous HOX9 genes are located in the most 3’ regions of the abdominal B-related gene regions in HOX complexes. Moreover, HOX9 genes are expressed specifically in the anterior portion of the developing limb buds.¹⁷ Therefore, HOX9 paralogsmight be involved in the development of the hip region. In 2009, Rouault et al²⁴ investigated the association of the CDH with two HOXB9 SNPs (rs8844 and rs2303486) in a sample population from France. The case-control study revealed no significant association between CDH and HOXB9 SNPs.²⁴ On the other hand, quite recently, Hao et al¹⁰ reported that rs2303486 was associated with CDH in a Chinese population. However, these studies did not evaluate a potential associated with AO. Human HOXB9 is located on the chromosome 17q21.3. HOXB9 cDNA is 2711 bp long with a 180 bp sequence element called the homeobox in exon2. HOXB9 encodes a 250 amino acid protein (NCBI Gene ID: 3219). Based upon genetic predisposition and the possible important function of HOX genes in the pathogenesis of skeletal disorders, it appears worthwhile to investigate whether HOXB9 gene polymorphisms are associated with susceptibility to AO.

In this case-control study, we identified the five SNPs (rs8844, rs3826541, rs3826540, rs7405887, rs79931349) located in the 3’ UTR of the HOXB9 gene that are associated with AO (rs 7405887 OR = 3.16, p = 5.29E-6, 95% CI 1.91 to 5.25; Table IV), and may be an independent risk factor for future development of AO. Interestingly, the two HOXB9 SNPs indicated OR < 1 respectively (rs2303485 OR = 0.524, p = 0.0091, 95% CI 0.322 to 0.855; rs2303486 OR = 0.519, p = 0.011, 95% CI 0.312 to 0.865) (Table IV). These two SNPs could be associated with decreased acetabular coverage. Lower acetabular coverage is a major and common cause of CDH, which is a contrasting disease to pincer-type FAI. The association of rs2303486 would be consistent with Hao et al’s report.¹⁰ Therefore, HOXB9 might be relevant to acetabular morphogenesis. However, the morphological mechanism of HOXB9 is not obvious, although HOX9 paralogs are critical during organogenesis, limb development and growth of tumour.^18,19,34,35 Further studies are needed to clarify the mechanism in detail that links the HOXB9 SNPs with the acetabular morphology. As shown in Table I, 32.3% of AO cases had AR (OR = 6.92, p = 1.87E-9, 95% CI 3.47 to 13.8). AR appears to be related to AO. AR is defined as a ‘crossover’ sign,^36,37 and is also thought to be the common cause of pincer-type FAI.^3,11,12 The HOXB9 SNPs may be associated with the pathogenesis of AR in the same way as AO.

This study had several limitations, such as a small sample size and a lack of replication study. Therefore, more studies should be conducted with larger sample numbers and using different ethnic groups. These future studies on HOXB9 function and its relevance to AO will contribute not only to the elucidation of the pathogenic mechanism of pincer-type FAI, but also to the identification of a novel therapeutic target for the disease. The HOXB9 SNPs could be a predictive tool to identify AO, helping to delay or prevent the onset of pincer-type FAI.