Association Between TPM1 Gene Polymorphisms and Idiopathic Congenital Talipes Equinovarus Risk in a Chinese Population

Introduction

Congenital talipes equinovarus (CTEV), or clubfoot, is a common congenital deformity of the lower limb in children (Matar et al., 2017; van Praag et al., 2018). The disease has an incidence of 0.15%, and it usually affects the right foot more often than the left foot (right: left = 2:1), while ∼50% of cases are bilateral (Chapman et al., 2000; Matar et al., 2017; Khan et al., 2020). The typical features of CTEV include forefoot adductus, hindfoot varus, and ankle equinus. Most cases of clubfoot (∼80%) are idiopathic CTEV (ICTEV), and ∼20% are related to neuromuscular diseases or arthrogryposis, spina bifida, and congenital syndromes (Chapman et al., 2000; Yang et al., 2016).

Although most patients achieve a very good initial correction after being treated by the Ponseti method, ∼54% of patients will experience recurrences (Hosseinzadeh et al., 2017, 2019; Gelfer et al., 2019; Thomas et al., 2019). The etiology of ICTEV is still unclear and understanding its cause may be relevant for reducing the recurrence rate and improving its treatment.

Family studies have shown that there is a high risk for first-degree relatives of affected children to also suffer from ICTEV (Lochmiller et al., 1998; Chapman et al., 2000; Yang et al., 2016; Basit and Khoshhal, 2018). Twin studies have shown that the risk of identical twins being affected is 32.5%, while for fraternal twins, it is only 2.9% (Barker et al., 2003; Heck et al., 2005; Engell et al., 2006). Therefore, genetic factors may play a fundamental role in the occurrence of ICTEV.

Clinical evidence shows that the foot length, calf girth, and thigh girth are all shorter on the affected side than on the normal side (Shimode et al., 2005). MRI scans have shown that the affected leg musculature is atrophied, and it does not improve as the patients grow (Ippolito et al., 2009, 2012; Duce et al., 2013). Histological and immunohistochemical findings showed that the myofibroblast-like cells seemed to suffer from a disorder of ligaments resembling fibromatosis, which may lead to abnormal contractions and result in ICTEV (Fukuhara et al., 1994). These findings suggest that abnormal muscle development may be involved in the development of ICTEV.

The tropomyosin gene 1 (TPM1) regulates the development of fast twitching muscle fibers, which are fast, but fatigue easily, while the tropomyosin gene 2 (TPM2) guides the development of slow twitching muscle fibers, which are slow, but long contracting. Weymouth et al. identified associations between rs4075583 in the TPM1 gene and ICTEV in a non-Hispanic white population (Weymouth et al., 2011). Moreover, single nucleotide polymorphisms (SNPs) in TPM2 were also found to be associated with distal arthrogryposis, which usually includes typical clubfoot features. However, TPM2 was found to be normal in clubfoot patients by using exome sequencing (Gurnett et al., 2009).

Rs4075583 is located in intron 1 of TPM1, which is a regulatory region. Rs2025126 is located upstream of TPM2, and rs2145925 is located in the first intron of TPM2. These three promoter variants in TPM1 and TPM2 combined with promoter variants of HOXA9 may eliminate nuclear protein interactions, which could decrease the activity of their promoters. This may be one mechanism involved in the occurrence of ICTEV (Weymouth et al., 2016). Moreover, genetic differences may explain its low incidence (0.57 of 1000) in Chinese populations compared to other populations (Werler et al., 2013).

In this context, we conducted a case-control study of 430 ICTEV cases and 891 ICTEV-free controls to explore the associations among these three potential polymorphisms and ICTEV susceptibility in a southern Chinese population.

Subjects and Methods

Results

Discussion

In this hospital-based case-control study, 430 patients with ICTEV and 891 controls were included. The associations of TPM1/rs4075583 G>A, TPM2/rs2145925 C>T, and TPM2/rs2025126 G > A polymorphisms with the risk of ICTEV were evaluated. The statistical results indicated that the A allele of TPM1/rs4075583 is associated with an increased ICTEV risk in this southern Chinese population.

Tropomyosin is an α-helical coiled-coil protein dimer that plays an important role in regulating muscle contraction. Usually, there are 4 TPM genes (TPM1, TPM2, TPM3, and TPM4) and their various isoforms, and these isoforms arise from each gene by using two alternative promoters and the differential splicing of exons 2, 6, and 9 (Janco et al., 2016). TPM1 regulates the development of quick, easily fatigued, fast-twitch muscle fibers. Katelyn found that the TPM1/rs4075583 polymorphism was associated with ICTEV in a non-Hispanic white population (Weymouth et al., 2011). This alteration of TPM1 promoter activity may cause abnormal expression of TPM1, which could lead to a longer duration of the attached state of myosin to the thin filament (Kopylova et al., 2019; Carlus et al., 2020). As a result, prolonged muscle contraction of the leg leads to foot contracture, characterized by clubfoot. Our study confirmed this association in a Chinese population.

TPM1/rs4075583 can create an allele-specific nuclear protein interaction, but this interaction is not sufficient to alter the activity of the TPM1 promoter. However, rs4075583/TPM1 can alter TPM1 promoter activity in the context of the surrounding ∼1.7 kb genomic architecture (Gurnett et al., 2009; Weymouth et al., 2011). In this study, the statistical results showed that TPM1/rs4075583 polymorphism had no significant association with clubfoot (p = 0.162) in the dominant model of A. These findings suggested that rs4075583/TPM1 may interact with other genes to affect muscle morphogenesis and/or contractions during the development of ICTEV. This hypothesis is consistent with the multifactorial inheritance model proposed for ICTEV (Lochmiller et al., 1998; Gurnett et al., 2007; Ester et al., 2009).

TPM2 has an important role in regulating the development of slow, long contracting muscles. Two studies found that rs2025126/TPM2 and rs2145925/TPM2 could create allele-specific nuclear protein interactions that individually had significant functional consequences by affecting promoter activity in muscle cells, which may be risk factors for isolated clubfoot (Ester et al., 2009; Weymouth et al., 2011). However, in our study, we found that neither of these two SNPs was associated with the risk of ICTEV. The rs2025126 SNP is located upstream of the TPM2 gene and can eliminate nuclear protein interactions when the alternate allele is present, so it can decrease the promoter activity of the TPM2 gene. As a result, decreased TPM2 leads to continuous actin-myosin interactions that cause perpetual muscle contraction, and a contracted foot is formed. In contrast, the rs2145925 SNP is located in the first intron of the TPM2 gene and can create a nuclear protein interaction to increase promoter activity when the alternate allele is present (Janco et al., 2016; Weymouth et al., 2016). The increased expression of TPM2 could lead to actin inhibition, which restricts muscle contraction and foot movement. This restriction potentially causes muscle wasting, manifested as calf muscle hypoplasia. The imbalance of these major muscular functions could contribute to the occurrence of clubfoot (Weymouth et al., 2016). Thus, we may need larger studies or additional mechanistic studies to fully evaluate the effects of TPM2 on the development of ICETV.

Our study explored the associations of TPM gene polymorphisms with ICETV risk in a southern Chinese population. However, there are several limitations of this study. First, the sample size is relatively small, inevitably reducing the statistical power. The power for rs4075583 was 0.5, which is relatively small. A larger sample size may be needed to increase the power, although the corrected p-value of the recessive mode of rs4075583 was 0.0355, which is less than 0.05. Second, the patients were from 4 different hospitals, while the controls were from only one hospital. This may cause selection bias, although all of the hospitals are located in southern China. Third, we did not evaluate environmental factors such as parental exposures and dietary intakes in this study, which may reduce the comprehensiveness of its analysis. Fourth, we performed a hospital-based case-control study to investigate the association between TPM polymorphisms and ICETV risk, which could not provide mechanistic information as to its etiology. The potential mechanisms need to be explored in cell lines and animal models in the future. Fifth, we included only the most frequently investigated TPM polymorphisms in this study, and other closely related TPM genes and polymorphisms were not examined. Finally, in our study, we used only the TaqMan real-time PCR method to genotype the SNPs. Next-generation sequencing (NGS) technology has made it possible to associate a clinical phenotype with overall changes in the genetic material (Basit, 2017). Moreover, it could sequence the complete human genome in a much shorter time than Sanger sequencing. Therefore, sequencing the complete human genome of patients with clubfoot by using NGS may yield interesting insights into its etiology.

Our study confirmed the hypothesis that ICETV is caused by muscular dysplasia. The development of muscular dysplasia is multifactorial, including the activity of various TPM genes. Because muscular dysplasia is present in the calf at birth in ICETV patients, muscular dysplasia associated with ICETV first develops in the embryo. Thus, the etiology of ICETV can only be explored by examining muscular dysplasia in embryos.