ICAM1 K469E Polymorphism Effect in Gastroschisis Patients

Gastroschisis (MIM No. 230750) is a congenital disorder characterized by fetal intestines' extrusion outside the body. The prevalence rate of gastroschisis varies among ethnic groups: 5 to 40 per 100,000 live births.¹ Its prevalence has been increasing in the United States and worldwide.²

There are several hypotheses about the pathogenesis of gastroschisis, including one involving an impairment in the normal attachment between umbilical cord and umbilical ring.³ This anatomic defect might be due to a reduced cell deposition and impaired cord attachment at the umbilical ring.³

Recently, several studies have demonstrated an association between gastroschisis risk and common variants of the cell-cell interaction gene intracellular adhesion molecule 1 (ICAM1).^4,5 So far, there have been no such reports from the Asian region. Therefore, an additional study is warranted in Asia, particularly in Indonesia, because the ICAM1 K469E polymorphism frequency differs among ethnic groups,⁶ and its effect on gastroschisis has never been determined to date. In this study, we analyzed the ICAM1 common variant K469E in the Indonesian population both to assess its control frequency and whether this polymorphism shows an association with gastroschisis.

Patients and Methods

DNA isolation and genotyping

Genomic DNA was extracted from abdominal wall tissue and/or a blood sample of the 48 gastroschisis probands and from the peripheral blood of the 88 control samples, using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The ICAM1 K469E polymorphism was chosen based on previous evidence.⁴ Genotyping of the variant was performed using the polymerase chain reaction–restriction fragment-length polymorphisms (PCR-RFLP) technique (Fig. 1). In brief, the ICAM1 fragment containing the K469E variant was amplified using forward primer 5′-CCATCGGGGAATCAGTG-3′ and reverse primer 5′-ACAGAGCACATTCACGGTC-3′. Subsequently, the PCR product was digested by endonuclease BstUI restriction enzyme. The E469 allele creates a restriction site for the enzyme.⁷ The digestion products were separated on 3% agarose gel and subsequently were visualized by ethidium bromide staining.

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Results

The frequencies of genotypes for ICAM1 K469E in patients were KK, 30 of 48 (63%); KE, 17 of 48 (35%); and EE, 1 of 48 (2%), whereas their frequencies in controls were KK, 48 of 88 (55%); KE, 33 of 88 (37%); and EE, 7 of 88 (8%). Those frequencies were not significantly different between both groups (P = 0.37), with an OR of 1.39 (95% CI, 0.68–2.85). The K469 allele had a frequency of 80% (77 of 96) in patients and 73% (129 of 176) in controls, and the frequency in patients was not significantly higher (P = 0.20), with an OR of 1.48 (95% CI, 0.81–2.70; Table 1). Furthermore, those genotypes were in Hardy-Weinberg equilibrium (P = 0.69).

Table 1  The genotypes and allele frequencies of ICAM1 K469E polymorphism in infants with gastroschisis and controls^a

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Then, we compared the observed risk allele frequencies in Indonesian controls with those reported for the 1000 Genomes Project Asian ancestry controls.⁶ The ICAM1 K469 allele (0.73 versus 0.72) had a frequency similar to that in the 1000 Genomes Project Asian ancestry individuals. However, the ICAM1 K469E showed high variation within Asia, with a range of 0.56 to 0.78 in Japanese and Southern Han Chinese.⁶

Moreover, the K469 allele had a frequency of 78% (47 of 60) in patients with maternal age younger than 25 years and 83% (30 of 36) in patients with maternal age 25 years or older, and the frequency in gastroschisis infants with maternal age younger than 25 years was not significantly higher (P = 0.55), with an OR of 0.72 (95% CI, 0.25–2.11; Table 2).

Table 2  The genotypes and allele frequencies of ICAM1 K469E in gastroschisis infants with regard to maternal age ^a

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Discussion

In this study, we have analyzed the frequency of the ICAM1 common variant K469E in an Indonesian population and its associations with gastroschisis. We were unable to find evidence of the genetic effect of ICAM1 K469E in Indonesian gastroschisis cases. To the best of our knowledge, this is the first study for the association between the ICAM1 common variant K469E and gastroschisis risk. Our study revealed that ICAM1 K469E is not a genetic risk for gastroschisis, with a background allele frequency of approximately 73% in Indonesia (Table 1), differing from a previous study.⁴ These differences might relate to Indonesian genetic structure ethnicity.⁹ Ojeda-Ojeda et al¹⁰ demonstrated that ICAM1 common variant K469E was strongly associated with polycystic ovary syndrome in Spain, but it was not a risk factor for polycystic ovary syndrome in other ethnic groups.¹¹

Furthermore, our recent study is another example of differential association of common variants with a disease in different ethnic backgrounds, an example being MTHFR polymorphism c.677C>T.¹² This variant has been associated with gastroschisis in our population, but it has not shown an effect on gastroschisis in white individuals.¹³ Other examples are NRG1 common variants rs16879552 and rs7835688. These polymorphisms have been related to Hirschsprung disease in Asian ancestry cases^14–16; however, they are not associated with Hirschsprung disease in white ancestry patients.^17,18 In contrast, SEMA3 rs11766001 polymorphism is strongly associated with Hirschsprung disease risk by case-control and TDT analyses in white individuals,¹⁷ but its frequency is almost absent in the Indonesian population.¹⁹

Interestingly, there was racial variability in the outcomes of neonates with gastroschisis.²⁰ Hispanic gastroschisis neonates showed significantly higher mortality than African American and white infants.²⁰ Further investigation is necessary to elucidate whether the outcomes of gastroschisis patients are affected by the common variant.

It has been shown that maternal age is a risk factor for gastroschisis.²¹ However, our study also showed that the frequency of ICAM1 K469 allele does not differ between the patients with maternal age younger than 25 years and the patients with maternal age 25 years and older (Table 2).

ICAM1 has been shown to have a role in the leukocytes' and monocytes' adhesion to the activated endothelium. ICAM1 has 5 extracellular domains involving circulatory leukocyte binding sites for recruiting it at the inflammation sites, and it has a tight adhesion with vascular endothelium to form macrophages transformed foam cells.²² Previous reports revealed the effect of ICAM1 polymorphism on gastroschisis.^4,5 However, our study was unable to show an association between ICAM1 K469E polymorphism and gastroschisis risk, implying that the gastroschisis pathogenesis might be affected by several genetic risks, not only ICAM1 K469E variant. Furthermore, it seems likely that the gene-environment interaction has more impact on gastroschisis pathogenesis than a single genetic etiology.⁵ Other concerns regarding our study were statistical power issues and small sample size.

Conclusions

ICAM1 K469E is not a common susceptibility factor for gastroschisis in Indonesia. A multicenter study with a larger number of participants is necessary to clarify these results.