Chitotriosidase, a prematurely orphan enzyme

HUMANEVOLUTION

Malaguarnera L. Department of Biomedical Sciences, University of Catania, Italy

Barone R. Institute of Neurological Sciences Catania Section, CNR, Cosenza, Italy

Angius A. Institute of Population Genetic, CNR, Alghero (SS), Italy

Musumeci S. Department of Pharmacology, Ginecology and Obstetrics, Pediatrics, University of Sassari and Institute of Population Genetic, CNR, Alghero (SS), Italy

Keywords: Chitotriosidase Polymorphism, Sub-Saharan area, Mediterranean area.

Vol.19- n. 1 (71-75)- 2004

Chitotriosidase, a prematurely orphan enzyme. Human Chitotriosidase (CHIT) is a member of the chitinase family and is synthesized by activated macrophages. Recently, a genetic polymorphism was found to be responsible for the common deficiency in CHIT activity, frequently encountered in different population. We analyzed the CHIT gene in some ethnic groups from Mediterranean and African area, to evaluate whether the CHIT gene polymorphism correlates with the changes in environmental features and the disappearance of parasitic diseases. We evaluate the plasma CHIT activity and analyzed, by PCR, the Chit gene polymorphism in 100 Sicilian, 107 Sardinian and 99 Sub-Saharan subjects. We found an heterozygote frequency for a duplication of 24 base pair in ex6n 10 of 44,54 % in Sicily and 32,71 % in Sardinia, whereas homozygous Chit deficient were 5,45 % and 3,73 %, respectively. On the contrary in Burkina Faso, a mesoendemic region for Plasmodium falciparum malaria and other intestinal parasites, a low incidence of CHIT mutation was found (heterozygous 2%) and any subject was homozygous for CHIT deficiency. Our results suggest that in sub-Saharan population the intact CHIT gene seems essential for sustaining resistance against chitin-coated parasitic disease, whereas the presence of CHIT gene. polymorphism in Mediterranean population could be the result of a recent positive selection due to improved environmental conditions, which makes prematurely orphan this enzyme.

Human chitotriosidase (Chit) belongs to the chitinase family, a group of enzymes with the capacity to hydrolyze chitin, synthesized by macrophages. The CHIT gene is localised in the chromosome lq31-q32 and consists of 12 exons and spans about 20 kb. The nature of congenital deficiency in Chit activity has been identified in a duplication of 24 base pair in exon 10, which results in activation of a cryptic 3' splice site, generating a m R N A with an in-frame deletion of 87 nucleotides (1). All Chit deficient individuals are homozygous for the duplication. We found by PCR reactions, using specific primers, an heterozygote frequency for a duplication of 24 base pair in exon 10 of 44,54 % in Sicily and 32,71% in Sardinia, whereas homozygous Chit deficient were

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5,45 % and 3,73 %, respectively. The presence of heterozygotes in individuals from various ethnic group from Europe to Indonesia (about 35%), demonstrates that this mutation is relatively old (1-2). Given the high incidence of Chit deficiency, it could be argued that this enzyme became redundant in human. A study of Choi et al (3) in South Indians demonstrates an association between Chit deficiency and susceptibility to Wuchereria bancroftfilaria infection (P = 0.013), confirming the important role of Chit in the infection by pathogens containing chitin. The role of chitinases against fungal pathogens in plants have been demonstrated and the features of this hydrolase are well conserved in the human Chit (4). Recent evidences suggest that this enzyme is increased also in conditions where the immune system is activated, like thalassemia syndromes (5). To obtain more insight on the role of Chit in other type of infections, we though interesting also determine whether Chit activity is increased in a condition, such as in Plasmodium malaria infection, where the immune system is activated by the presence of specific parasitic product into phagocytic cells. Our study on the levels of this enzyme in children with malaria demonstrated that it is regulated in a singular manner. Chit levels were found higher (median 299 nmol/ml/h; range 188-521) in children with malaria than to African control children (median 63 nmol/ml/h; range 4-450) (P < 0.0001) (5). Therefore, the high plasmatic levels of Chit in children affected by malaria represent a reactant of acute phase of malaria conf'Lrrning that the level of chitotriosidase represents an important marker of disease activity. The severity and the fatal outcome of the disease could be the results of an abnormal macrophage activation, which may be expressed by the Chit level. In fact the bimodal distribution of chitotriosidase levels could have a critical role, as well as some cytokines in the adaptive immune response to malaria through macrophage activation, directly related to the effects of hemozoin (HE) on phagocytic cells (6). These observations lead to deduce that Chit is phylogenetically very old as confirmed by the primitive role of macrophage in the defence mechanism against protozoa and helmints. But the evolutionary consequence of the mutation in the CHIT gene could be the loss of its original action against chitincoated micro-organisms and parasites to gain the capability of specific intracellular signal in the immune activation. Only the study of immune response in vivo and in vitro in relation to the mutation of CHIT gene will clarify the role of this enzyme in immunity. Remain intriguing to understand why the mutation of CHIT gene maintains its high incidence in different ethnic groups, thus questioning the selective advantage for carders of the mutation, but, at the present, there are no clues for the functions of such selective factors. However the observation of a very low incidence (2%) of Chit deficiency in black population from a Sub-Saharian area with high endemicity for malaria and infections

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sustained by chitin-containing pathogens like nematodes (7), show that the persistence of parasitic diseases may have preserved the wild chitotriosidase gene in such regions. The gene frequency of wild type and mutant alleles and the Chit activity levels in different populations from Mediterranean to Sub-saharan areas are shown in Table 1-2. Finally, it is interesting to note that in the populations of Mediterranean area, (where the malaria infection was eradicated after the II World War) the Chit levels are lower than Sub-Saharan population. Moreover, the little differences occurring between Sicilian and Sardinian (8) (median 37.5 range 6-100 nmol/ml/h and median 42 range 4222 nmol/ml/h respectively), could be related to the diversity in some environmental factors between the two populations. Sardinian subjects come from a geographical area with a past intermediate malarial endemicity in comparison to East Sicily where malaria was less endemic, and this is confirmed by the elevated frequency of B-thalassemia trait and G6PD deficiency in Sardinia (9, 10). Of note is a phylogenetic overlap between Chit and B-thalassemia: the presence of Plasmodium falciparum has advantaged the 13-thalassemia gene, while chitin containing pathogens have preserved the wild CHIT gene. In fact, our previous studies in Sicilian and Sardinian patients with f~-thalassemia, have demonstrated that the level of Chit in Sardinian (Thalassemic patients) was higher than in Sicilian (Thalassemic patients) and this is in accordance with the different percentage of mutated alleles found among the two populations. In conclusion our results give evidence that the presence of wild CHIT gene or the absence of mutation could represent a protective factor in the ethnic groups in which disadvantageous environmental conditions and parasitic diseases persist up today. Nevertheless, we can not rule out the possibility that the loss of the original action of this enzyme against chitin-coated parasites, as the evolutionary consequence of the parasite disappearance, may represent a requisite to gain other important physiological roles in the immune response (11). However, the possibility that the absence of a well defined pressure which might confer some advantage to the heterozygote carder of chitotriosidase mutation warrants further investigation. Suitable detections of CHIT gene mutation frequency in different ethnic groups, including conditions where the macrophages are activated, might add new information, into the physiological role of this enzyme, became orphan prematurely.

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Table 1. Frequencies of wild-type and mutant alleles in Sicilian, Sardinian and African samples.

Number of subjects Homozygous wild type (wt) Heterozygous wt/mutant Homozygous mutant

Sicilian 100 51.01% 44.54 % 5.45 %

Sardinian 107 63.56% 32.71% 3.73 %

African 99 98% 2% 0 %

Table 2. Chitotriosidase activity (nmol/ml/h) in Sicilian subjects.

Homozygous wild type (wt) Heterozygous wt/mutant Homozygous mutant

Mean (SD) 46.87 (16.20) 29.27 (17.60) 1.21 (1.07)

Median (range) 44 (6-73) 24 (4-102) 2 (0-2.07)

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Received: December 29, 2002

Accepted: June 10, 2003