Association of PFKM gene polymorphisms and susceptibility to cryptorchidism in a Chinese Han population

Cryptorchidism is one of the most common congenital anomalies in newborn boys. There are various risk factors that have been verified to have relationship with cryptorchidism, including exogenous and genetic, but the pathogenesis of cryptorchidism remains unclear. PFKM gene is a critical gene encodes for a regulatory enzyme, which limits the rate in the pathway of glycolysis. We assumed that cryptorchidism risk may associated with PFKM gene single-nucleotide polymorphisms (SNPs). Thus we selected three tag SNPs in the PFKM gene and aimed to investigate the possible association between PFKM gene polymorphisms and cryptorchidism risk. The SNPs were genotyped using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. 140 cases and 227 controls were enrolled in this study, including 105 unilateral cryptorchidism and 35 bilateral cases. The testis position was decided by the higher one in bilateral cases. The frequency of allele G of SNP rs2228500 is increased in cryptorchidism patients compared to that in controls (p < 0.05). Genotypic frequencies of rs2228500 are associated with the susceptibility of cryptorchidism in the codominant model (p < 0.05). And compared with G/G genotype in the dominant model, notable decreased frequencies of A carriers (A/G–A/A genotypes) were observed in cryptorchidism patients (p = 0.0069, OR = 1.80, 95% CI  1.17–2.75). This research first revealed that PFKM gene polymorphisms were associated with cryptorchidism in a Chinese Han population. We have offered primary evidence that the G allele and the G/G genotype of rs2228500 SNP in the PFKM gene are more frequent in patients with cryptorchidism than healthy controls.


Introduction
Cryptorchidism is a common congenital malformation in newborn boys, affecting 3-5% of the full-term-born male infants and up to 30% of preterm or low birth-weight male neonates [1]. It is a clinical finding in which one or both testicles fail to descend into the ipsilateral scrotum, also known as undescended testis (UDT). Sufficient evidence suggested that cryptorchidism (especially bilateral UDT) is one of the prevalent causes of male infertility, and it is also a higher risk factor for germ cell tumors [2,3]. At present, the surgical operation is the first treatment for most patients with cryptorchidism, and the orchiopexy should be performed before 18 months age as far as possible to preserve the fertility of children and reduce the risk of testicular cancer in the future [4,5].
Typically, completely descended testes (CDT) is formed through a distinct, sequential two-stage descent, each involving multiple mechanical and hormonal factors [6]. The complexity of the biological mechanisms driving testicular descent is probably a vital factor in the complexity of the etiology of the disease. However, pieces of evidence proved environment and genetic factors may both play critical roles in the development of cryptorchidism [7]. In recent years, there has been an increasing interest in research on the correlation between cryptorchidism and pathogenic genes or susceptibility genes. Some researchers believe that cryptorchidism caused by different pathogenic genes may be accompanied by different symptoms and prognosis, so personalized treatment should be adopted. Therefore, the discovery of new cryptorchidism pathogenic genes or susceptibility genes is of great significance for molecular diagnosis, treatment selection, disease prognosis prediction, and prenatal consultation.
The PFKM gene is located within a 41 kb region at chromosome 12q13.11. PFKM is the encoding gene for phosphofructokinase-M, which plays an important role in energy metabolism and muscle cell homeostasis. Phosphofructokinase-M is one of the key speed-limiting enzymes for glycolysis, and it is involved in the energy metabolism pathway through glycolysis [8]. In human cells, phosphofructokinase (PFK) is a tetrameric enzyme that is subjected to allosteric regulation. PFK consists of three isozyme types, which are liver-type (PFKL), platelet-type (PFKP), and muscle-type (PFKM). Tissue isozymes randomly aggregate to form homotetramers or heterotetramers depending on the relative abundance of the subunits in a particular tissue. PFKM is abundant mainly in skeletal muscles as a homotetramer and is expressed as a heterotetramer in erythrocytes and various other tissues [9]. In recent years, PFKM has attracted extensive attention in tumor research and many studies have shown that it is highly correlated with tumor cell growth [10][11][12]. Earlier in vivo studies have shown that PFK showed high activity in the rat testis during maturation [13,14]. Furthermore, PFK activity is decreased in the testis in the early cryptorchidism [13]. The remarkable thing is a significant muscle movement is required during testicular descent [15]. Hence, PFKM may participate in the normal growth and development of testis and testicular diseases.
We speculated that the PFKM gene may associate with cryptorchidism risk. To reveal whether PFKM is involved in the development of cryptorchidism, we conducted a case-control study to assess the role of 3 tag SNPs of PFKM (rs2228500, rs4075913, and rs11168417) in patients with cryptorchidism. To our knowledge, this is the first study to evaluate the correlation between PFKM gene polymorphisms and cryptorchidism risk.

Study subjects
The retrospective study was approved by the Ethics Committee of the West China Hospital and written informed consent was obtained from all participants. 140 patients with cryptorchidism and 227 unrelated healthy volunteers were enrolled in this study, and all the patients were recruited from the West China Hospital from February 2017 to March 2020. The boys were examined shortly after birth and again at 3 months of age to determine. The examination technique and the definition of cryptorchidism developed by Scorer [16] were applied. All examinations for these children were performed in a supine position under warm conditions. Testicle position was recorded after manipulation of the testis to the farthest distal position along the pathway of normal descent using firm and non-forced traction. Re-confirmation of the diagnosis before orchidopexy was conducted when the patient is around 1 year old. 105 cases manifested unilateral cryptorchidism and 35 cases were bilateral cryptorchidism in the case group. The testis position was decided by the higher one in bilateral cases. Among patients, the testis of 46 cases was in the superficial inguinal pouch while 34 cases in the pre-scrotal region, 31 cases in the external ring, 19 cases in the inguinal canal, and 10 cases in the internal ring. The subjects in the control group (mean age 4.8 ± 1.2 years, range 1 month-14 years) were admitted for resection of foreskin. For individuals in both groups, premature or low birth-weight infants and any personal or family history of cryptorchidism or serious disease were excluded to avoid multifactorial effects. All the subjects were Chinese Han population living in the Sichuan Province of southwest China.

SNP selection and genotyping
Tag SNPs are representative SNPs in genome regions with high linkage disequilibrium. Three tag SNPs of the PFKM gene were selected out according to the data from tag SNPs genotyped in the CHB (Han Chinese in Beijing, China) population sample of the international HapMap project (http:// www. hapmap. org/ index. html. zh).
DNA isolation kit from BioTeke (Peking, China) was used to extract genomic DNA from each individual's blood sample according to the manufacturer's instructions and the DNA products were stored at -20 ℃ for further genotype. The rs2228500 (A/G), rs4075913 (A/G), and rs11168417 (C/T) SNPs in the PFKM gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Primers for PCR were designed by the online software Primer3.0. The primer sequences and the information about restriction enzymes for digesting PCR products are shown in Table 1. About 10% of the samples were randomly selected for repeated genotyping, thus the results were 100% consistent.

Statistical analysis
SPSS ver. 26.0 (SPSS Inc., Chicago, IL, USA) was applied to analyze all the data. The allele and genotype frequencies of the three selected SNPs were obtained through directed computing and the Hardy-Weinberg equilibrium was evaluated by chi-squared test. The genotype associations between the PFKM gene and cryptorchidism susceptibility were calculated by SNPstats online analysis software (https:// www. snpst ats. net/ start. htm), which assessed the frequency distributions within four genetic models (codominant, dominant, recessive, and overdominant) in cryptorchidism patients and healthy controls. Odds ratio (OR) and respective 95% confidence interval (95%CI) were reported to evaluate the statistical differences between alleles and genotypes. P value < 0.05 was considered as statistically significant.

Results
Three tag SNPs were genotyped in 140 cryptorchidism patients and 227 healthy control subjects. The genotype distributions of these polymorphisms all conformed to the Hardy-Weinberg equilibrium. The allele frequencies of the PFKM tag SNPs in patients with cryptorchidism and control individuals are summarized in Table 2. The allele frequencies of rs4075913 and rs11168417 polymorphisms have no significant differences between cryptorchidism patients and healthy controls. However, the frequency of the G allele of the rs2228500 in patients with cryptorchidism is significantly higher than those in controls (78% vs.69%). By contrast, the A allele frequencies of rs2228500 decreased (22% vs.31%) in the case group. A significantly increased cryptorchidism risk was found to be associated with the G allele of the rs2228500 (p = 0.010, OR = 1.58, 95% CI 1.11-2.24).
As shown in Table 3, the genotype distributions of G/G, A/G, and A/A of rs2228500 were 60.7%, 33.6%, and 5.7% in the case group, and 46.3%, 44.9%, and 8.8% in the control group, respectively. Obviously, the significance could be observed in the codominant model (p = 0.025). In the dominant model, compared with the G/G genotype, A/G-A/A genotypes were associated with a significantly decreased risk of cryptorchidism (p = 0.0069, OR = 1.80, 95% CI 1.17-2.75). No significant correlation was observed in any genetic models of rs4075913 and rs11168417 polymorphisms with the risk of cryptorchidism.

Discussion
Cryptorchidism refers to undescended testis, is one of the most frequent urogenital abnormalities in newborn boys [17], and it is the best-characterized risk factor for reduced fertility in males and testicular neoplasia [7]. In humans, testosterone and insulin-like factor 3 (INSL3) are the main regulators for testicular descending from the abdominal cavity to the bottom of the scrotum [18,19]. Mutations in the genes  encode for INSL3 and its receptor and androgen receptors were considered to be the main cause of some forms of cryptorchidism [20][21][22]. Although some cases of cryptorchidism in humans can be attributed to known genetic defects and several pathogenetic mechanisms for cryptorchidism have been described, the exact cause of cryptorchidism in most patients remains unknown. PFKM gene maps on chromosome 12q13.11 and its coding region consist of 2340 bp nucleotides, which encodes approximately 780 amino acids [23]. A key glycolytic regulatory enzyme, PFKM, which is concerned as an energy activator of muscle glycolysis, is encoded by the PFKM gene. Recently, there have been several researches focused on the association between PFKM mutations and different types of cancers, such as bladder cancer [24], breast cancer [25], and ovarian cancer [26], etc. Although some studies have confirmed the existence of specific PFKM in testis, there is no study on the correlation between PFKM gene and cryptorchidism. What is even more interesting is that earlier research in rat embryos indicated that growth retardation and congenital defects could be caused by interfering with glycolysis, which is an important source of ATP production [27]. As is known to us, PFKM is a pivotal regulator of cellular glycolysis by catalyzing the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. PFKM deficiency is characterized by muscle weakness due to fuel crisis in exercising muscles [28]. Taken together, there may be a significant role of the PFKM gene plays in the pathogenesis of cryptorchidism. Among the three tag SNPs we have chosen in PFKM, one SNP is located in the intron region and was observed to be associated with cryptorchidism risk, and the other two SNPs are located in the synonymous codon regions and both of them have no significant correlation with cryptorchidism risk. Julia et al. [29] identified that muscle patterning defection is associated with cryptorchidism in the rat fetal. Therefore, we suspected that the tag SNP rs2228500 may be affecting protein structure by influencing coding amino acids. In addition, it may affect the energy metabolism in the muscle of the glycolysis pathway, which may be a key factor in the lack of testicular descent motivation.
In summary, we investigated the impact of the PFKM gene polymorphism on cryptorchidism, and we observed significant differences in the frequency of alleles and genotypes at 1 tag SNP between patients and controls. We have offered primary evidence that the G allele and the G/G genotype of rs2228500 SNP in the PFKM gene are more frequent in patients with cryptorchidism than healthy controls. It implies that the polymorphism of the PFKM gene locus (rs2228500) may be a new genetic marker for cryptorchidism susceptibility and these alleles and genotypes may be risk factors for this disease.
Although we detected the association between the PFKM SNPs and cryptorchidism, there were certain limitations in this study. The sample size and ethnic types of this study were small and these results need to be further confirmed in a larger cohort. Moreover, the function and the underlying signal transduction mechanisms of the PFKM gene in cryptorchidism development need to be clarified.