A Polymorphism in the Chitotriosidase Gene Associated with Risk of Mycetoma Due to Madurella mycetomatis Mycetoma--A Retrospective Study

Abstract

Background: Madurella mycetomatis is the most prevalent causative agent of eumycetoma in Sudan, an infection characterized by the formation of grains. Many patients are exposed to the causative agent, however only a small number develop infection. M. mycetomatis contains chitin in its cell wall, which can trigger the human immune system. Polymorphisms in the genes encoding for the chitin-degrading enzymes chitotriosidase and AMCase were described, resulting in altered chitinase activity. We investigated the association between 4 of these polymorphisms and the incidence of M. mycetomatis mycetoma in a Sudanese population.

Methodology: Polymorphisms studied in 112 eumycetoma patients and 103 matched controls included a 24-bp insertion in the chitotriosidase gene (rs3831317), resulting in impaired chitinase activity and single nucleotide polymorphism (SNP) in the AMCase gene (rs61756687), resulting in decreased AMCase activity. Also, a SNP (rs41282492) and a 10-bp insertion in the 5'UTR region of the AMCase gene (rs143789088) were studied, both resulting in increased AMCase activity. DNA was isolated from blood and genotypes were determined using PCR-RFLP.

Principal findings: Histological staining proved the presence of chitin in the fungal grain. The polymorphism resulting in decreased chitotriosidase activity was associated with increased odds of eumycetoma (odds ratio 2.9; p = 0.004). No association was found for the polymorphisms in the genes for AMCase (all p>0.05).

Conclusion: Decreased chitotriosidase activity was associated with increased risk of M. mycetomatis mycetoma.

Fig 1. Tissue sections of Mycetoma foot, showing the fungal grain.

Magnification 400x. (A) Haematoxylin and eosin staining. The grain, consisting of cement and fungal hyphae, is colored red. Around the grain, a zone with neutrophils is visible. (B) Grocott’s methenamine silver staining. The hyphae inside the grain are stained black. (C) Calcofluor white staining. Chitin is stained by calcofluor white staining [18]. This photo illustrates that hyphae inside the grain are stained, and not the cement component of the grain.

Fig 2. Tissue sections of Mycetoma foot stained for chitotriosidase and for AMCase.

Magnification 100x (A and C) and 400x (B and D). (A) and (B): Chitotriosidase. (C) and (D): AMCase. Presence of both enzymes is shown by red color. The grain is clearly visible and colored red diffusely. Inside the grain, fungal hyphae are stained more intensely, showing an increased presence of chitotriosidase and AMCase around the fungal hyphae.