Preliminary evidence of an association between the functional c-kit rs6554199 polymorphism and achalasia in a Turkish population


Address for Correspondence
Yusuf Yilmaz, Department of Gastroenterology, Marmara University, School of Medicine, Pendik, Istanbul, Turkey.
Tel: +90 533 4403995; fax: +90 216 6886681;


Background  C-kit-positive interstitial cells of Cajal (ICC) of the lower esophageal sphincter are reduced in achalasia. Two functional gene polymorphisms (rs2237025 and rs6554199) within the c-kit gene may affect its transcriptional activity. In this pilot study, we hypothesized that these polymorphisms would be associated with achalasia.

Methods  Genomic DNA was extracted and real-time PCR reactions were used to determine the rs2237025 and rs6554199 c-kit polymorphisms in 88 Turkish patients with achalasia and 101 healthy controls.

Key Results  The frequency of the T allele of rs6554199 was significantly higher in patients with achalasia [odds ratio (OR): 1.55; 95% confidence interval (CI), 1.03–2.34; P = 0.038] compared with the G allele. Under a dominant model of inheritance, the carriage of at least one T allele was significantly more frequent in patients with achalasia (80.7%) than in controls (65.3%; OR: 2.21; 95% CI, 1.13–4.33; P = 0.022). No association of the c-kit rs2237025 polymorphism with achalasia was detected.

Conclusions & Inferences  Despite the small sample size and the possibility of a false positive finding, our preliminary data support the hypothesis that the T allele of the c-kit rs6554199 polymorphism may be associated with achalasia in the Turkish population. These findings need to be replicated in other racial-ethnically diverse populations.


Achalasia is an esophageal motor disorder characterized by incomplete relaxation of the lower esophageal sphincter (LES) and by the absence of esophageal peristalsis.1,2 Patients typically present with regurgitation, dysphagia, retrosternal pain, and marked weight loss. The most widely accepted hypothesis of the development of achalasia is that an initial insult to the esophagus, such as a viral infection, causes inflammation of the myenteric plexus.3 The etiological factors that trigger these inflammatory processes are not well understood, although genetic factors may play an important role.4

Interstitial cells of Cajal (ICC) have been shown to be involved in nitrergic neurotransmission of the LES.5,6 Evidence suggests that a reduction in the network of ICC in the esophageal wall is associated with impaired nitric oxide synthase expression in achalasia patients, which in turn can lead to the development of different gastrointestinal (GI) motor disorders.7–9 A transmembrane tyrosine kinase receptor (KIT), encoded by the protooncogene c-kit, is essential for the development and function of ICC.10,11 Of note, defects in the c-kit gene have been shown to cause GI motor disturbances in mice, mainly as a result of a loss of ICC.12 Two gene polymorphisms (rs2237025 and rs6554199) within the c-kit gene may affect its transcriptional activity.13,14 As an additional mechanism, and in view of the abundant expression of c-kit in ICC,10 in this pilot genetic association study we hypothesized that these polymorphisms may be associated with achalasia.

Materials and methods

Study participants

A total of 88 unrelated Turkish patients with achalasia (52 females and 36 males) who attended the Gastroenterology Unit of the Marmara University between September 2007 and June 2009 were included in this pilot study. The diagnosis was confirmed in all cases by esophageal manometry, absence of peristalsis in the esophageal body, and elevated intraesophageal pressures relative to the gastric baseline. The ethnically-, age-, and sex-matched control group included 101 healthy subjects (mainly blood donors and patients’ relatives without GI complaints).

The study was approved by the ethics committee of the Marmara University Medical School and performed according to good clinical practice guidelines. Written informed consent, which included subsequent genetic analyses, was obtained from all patients before study entry.

Selection of single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) within the genomic region of human c-kit gene were selected from the data set of International HapMap Project ( More than 400 SNPs described within the c-kit gene have been described, including variants affecting gene transcription. Using a validated tag SNP containing six polymorphisms identified using the SNP Browser 4.0 (Applied Biosystems; genotype correlation threshold 100%, minor allele frequency > 20%, Caucasian population), we selected two functional SNPs known to affect transcription, i.e., rs6554199 and rs2237025.13,14


Genomic DNA was isolated from peripheral blood leukocytes using the High Pure PCR Template Preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). Genotyping of the c-kit rs2237025 and rs6554199 polymorphisms was performed by real-time PCR and hybridization probe melting point analysis using a Light Cycler™ 1.5 instrument (Roche Diagnostics GmbH). The primers and hybridization probes for the rs6554199 polymorphism were designed using the GenBank reference sequence for c-kit (accession number NT_022853). The PCR was performed using a LightCycler® Fast Start DNA Master HybProbe Kit (Roche Diagnostics GmbH) in a final volume of 20 μL. The reaction mixture contained 1 × Fast Start DNA Master HybProbe, 1.5 mmol L−1 MgCl2, 0.3 μmol L−1 each primer, and 0.15 μmol L−1 hybridization probe. For the rs6554199 polymorphism, the sense primer was 5′-TTATCTGTCATCCTAAGAAAAACA-3′and the antisense primer was 5′-CCTCACAGTCCTTATAATCAAGT-3′. The sequences of the probes for the rs6554199 polymorphism were as follows: 5′-CACAGAGACCTTTTGTATTTCAGTTC-FL (donor) and LC640-CCTACAGCCGCATGACTCGAAC-p (acceptor). For the rs2237025 polymorphism, we used the primers and probes provided with a TIB MOLBIOL LightMix® kit (TIB MOLBIOL GmbH, Berlin, Germany). The sequences of primers and probes were not disclosed by the manufacturer.

The PCR conditions consisted of 95 °C for 10 min (for the initial denaturation phase) followed by 45 cycles of 95 °C for 5 s (denaturation), 55 °C for 20 s (annealing), and 72 °C for 20 (extension). Melting curve analysis was performed immediately after the amplification protocol under the following conditions: 0 s (hold time) at 95 °C, 30 s at 40 °C, and a slow up-ramp (0.1 °C s−1) for continuous acquisition to 85 °C.


The study power was calculated using the StatMate software, version 2.0 (GraphPad, San Diego, CA, USA). Based on the observed prevalence of the T allele of rs6554199, our sample size had a 75% power to detect a relative risk of 1.54 for achalasia between carriers and non-carriers with a significance level (alpha) of 0.05 (two-tailed). Chi-squared tests were used to test for deviation of genotype distributions from the Hardy-Weinberg equilibrium and for categorical variables. Normally distributed parameters were compared using a two-sample independent t-test. Allelic and genotypic distributions were estimated by allele counting and compared in the achalasia and control groups using the Fisher’s exact test. Crude odds ratios (ORs) are reported with their 95% confidence intervals (CIs). Analyses were performed using SPSS software (release 16.0; SPSS Inc., Chicago, IL, USA). A two-sided P value of < 0.05 was considered statistically significant. As this study was exploratory in nature as it merely provides a new hypothesis that requires confirmation or rejection by subsequent replication in other racial-ethnically diverse populations, a Bonferroni’s correction was not applied.


Table 1 shows the general characteristics of patients with achalasia. The mean LES pressure was 33.50 ± 16.15 mmHg and dysphagia was present in all patients.

Table 1.   General characteristics of patients with achalasia
 Males, n (%)36 (40.9)
 Females, n (%)52 (59.1)
Mean age, years
 Males49.0 ± 15.2
 Females49.4 ± 14.5
Dysphagia, n (%) 88 (100)
Esophageal regurgitation, n (%) 65 (74)
Weight loss, n (%) 48 (54.4)
Chest pain, n (%) 31 (35.2)
Heartburn, n (%) 16 (18.2)
Globus, n (%)17 (19.3)
Absence of peristalsis, n (%)88 (100)
Mean LES pressure (mmHg)33.50 ± 16.15

The observed genotype distribution for rs2237025 and rs6554199 polymorphisms did not deviate from Hardy-Weinberg equilibrium. The genotype and allele distribution of the rs6554199 c-kit polymorphism in patients with achalasia and healthy controls are reported in Table 2. The frequency of the T allele of rs6554199 was significantly higher in patients with achalasia (OR: 1.55; 95% CI, 1.03–2.34; P = 0.038) compared with the G allele. Under a dominant model of inheritance, the carriage of at least one T allele was significantly more frequent in patients with achalasia (80.7%) than in controls (65.3%; OR: 2.21; 95% CI, 1.13–4.33; P = 0.022). No association of the c-kit rs2237025 polymorphism with achalasia was detected (data not shown).

Table 2.   Genotype and allele distribution of the rs6554199 polymorphism in patients with achalasia (n = 88) and healthy controls (n = 101)
 Achalasia, n (%) Controls, n (%)P valueOdds ratio (95% CI)
  1. CI, confidence interval; ns, not significant.

 Codominant model
  GG17 (19.3)35 (34.7)  
  GT55 (62.5)54 (53.4)  
  TT16 (18.2)12 (11.9)ns 
 T dominant model
  TT plus GT71 (80.7)66 (65.3)  
  GG17 (19.3)35 (34.7)0.0222.22 (1.13–4.33)
  T87 (49.4)78 (38.6)  
  G89 (50.6)124 (61.4)0.0381.55 (1.03–2.34)


The transmembrane tyrosine kinase receptor c-kit is a 145–165-kD protooncogene that is structurally related to platelet-derived factor receptor and the colony stimulatory factor receptor.15 The primary ligand for c-kit is stem cell factor (SCF), which is also known as mast cell growth factor and steel factor.16 Previous studies have shown that genetic variants in the c-kit gene may alter the activity of the tyrosine kinase receptor, ultimately leading to several disease conditions including aplastic anemia,17 mastocytosis,18 and GI stromal tumors.19 Polymorphisms in the c-kit gene have been also investigated in relation to bone mineral density20 and male infertility.21 Growing evidence suggests that esophageal ICC express c-kit, which is required for their development and physiological activity.10 Importantly, alterations in ICC have been reported in congenital diseases with abnormal peristalsis.8,9 Starting from these premises, it seemed to be biologically plausible that functional variations in the c-kit gene could influence the genetic susceptibility to achalasia.

The results revealed that the rs6554199 but not rs2237025 c-kit polymorphism is associated with achalasia in a Turkish population sample. The rs6554199 polymorphism is a promoter variant (G>T) located 1694 bp upstream of the c-kit gene. It acts as a putative CCAAT enhancer-binding protein alpha (C/EBPα) binding site and can affect transcriptional activity.13 The rs2237025 polymorphism is an intronic variant (A>G) located at the 2881762 contig position in intron 1. It has been previously shown that this polymorphism may influence the alternative splicing of the gene and, in turn, gene expression.13 However, the mechanisms by which the T allele of the rs6554199 could affect susceptibility to achalasia remain to be determined. As ICC have a central place in research aimed at examining esophageal contractions and the etiology and pathogenesis of various motility disorders,5,6 we speculate that this polymorphism could influence the risk of developing achalasia by affecting the function and/or number of these cells. In any case, it should be observed that, whatever the mechanisms behind the association between the rs2237025 c-kit polymorphism and achalasia, our present results strongly support the emerging hypothesis that genetic factors may be involved in the pathophysiology of esophageal motor disorders.4

Our study has several strengths and limitations. The major strength of our study is that we have investigated a homogenous cohort of patients, and used the gold standard for the assessment of achalasia. The relatively small sample size of our study needs to be acknowledged as the major limitation. We are entirely aware that an isolated finding like the association between the minor T allele of rs6554199 and achalasia immediately raises the question as to whether we should allow for multiple testing, e.g., through the application of a Bonferroni’s correction, to deflate the perceived significance of the individual result and to avoid false positives. However, the tested polymorphisms covered the same given gene region (i.e., the c-kit gene). In this context, the conventional approaches, such as the Bonferroni’s correction, to correct for multiple testing are probably too stringent and may result in false negatives.22,23 Polymorphisms tested in complex diseases like achalasia rarely account for a large amount of variance, characterized by very low P values (P < 0.001). Thus, false negative is an important issue in this study and Bonferroni’s correction was not applied to the present data. In addition, our sample size was appropriately powered based upon the observed frequency of the minor T allele of rs6554199 in our population. Another important limitation of our study is its correlative nature, as we are not able to provide the mechanism underlying the association between the rs6554199 c-kit gene polymorphism and esophageal motor function. It is also possible that the observed association is caused by a gene variant that is in linkage disequilibrium with the examined polymorphism of the c-kit gene. Humanized’ gene targeted mice expressing different alleles of a gene of interest are increasingly used to study the functional effects of gene polymorphisms, and such an approach may help to clarify whether the rs6554199 c-kit gene polymorphism is the causal variant. Finally, the results of this study should be confirmed outside of Turkey, because ethnic differences are frequently reported in genetic association studies and we did not include a replication cohort.

Despite the small sample size and the possibility of a false positive finding, our preliminary data support the hypothesis that the T allele of the c-kit rs6554199 polymorphism may be associated with achalasia in the Turkish population. Our findings may add a novel risk factor to the multi-factorial genetic contribution to this rare esophageal motor disorder. Future investigations should be focused on understanding the exact role of c-kit genetic variants in influencing the number and distribution of ICC in achalasia.


This study was supported by grants from Scientific Research Project Coordinator Unit of Marmara University (Project No: SAG-C-TUP-171108-0253). All authors declare they have no actual or potential competing interests.

Author contribution

YOA designed the study and wrote the manuscript; FE designed the study, performed genetic analyses and helped to draft the manuscript; AG and OA visited the patients and performed all manometry; FG and AEK collected and analyzed the data; YY performed literature search and revised the manuscript critically; OK has made substantive intellectual contributions to the study; CK designed the research study and provided overall supervision.