High prevalence of chitotriosidase deficiency in Peruvian Amerindians exposed to chitin-bearing food and enteroparasites

Highlights • Catalytic deficiency of chitotriosidase has a very high frequency in Amerindians highly exposed to chitin from enteroparasites and diet.• Mutation frequencies are similar to those found in East Asian populations, and is probably conserved for a founder effect.• Such condition precludes the use of CHIT1 as a disease biomarker in South American populations with strong ethnic ancestry.


Introduction
Due to its unique biomechanical properties, chitin is one of the most abundant biopolymers in the biosphere (Muzzarelli et al., 2012;Musumeci & Paoletti, 2009) constituting structures with defensive (fungal cell wall, nematode egg shell), predatory (hooks) or nutritional (pharynx, mollusc radula) functions in many eukaryotic organisms, such as protozoans, insects and nematodes (Muzzarelli, 2011;Zakrzewski et al., 2014). While humans do not have the ability to synthesize chitin, they are known to produce chitinolytic enzymes. Chitotriosidase (CHIT1, or macrophage chitinase), together with acidic mammalian chitinase (AMCase) is one of the two known human enzymes able to cleave chitin. It is highly expressed by activated macrophages (Van Eijk et al., 2005) and is pre-formed in granules of neutrophils (Boussac & Garin, 2000). At the tissue level, CHIT1 is expressed in the human lung (Seibold et al., 2008), human lachrymal glands (Hall, Morroll, Tighe, Götz, & Falcone, 2008) and in both bone marrow and spleen of mice (Boot et al., 2005).
The expression in key innate immune cells and tissues at the host/environment interface is strongly suggestive of an involvement in innate immunity, for example against chitin-bearing pathogens such as fungi. Indeed such a protective function is well known to be an important element of immunity in plants, where chitinases are found amongst the so-called pathogenesis-related proteins (Kasprzewska, 2003). There is also increasing evidence for a role in innate immunity in mammals, particularly against fungal pathogens. Indeed, CHIT1 inhibits pathogenic chitin-producing fungi including Candida albicans (Vandevenne et al., 2011), Aspergillus niger and Cryptococcus neoformans (Gordon-Thomson et al., 2009), though it was concluded that it is less effective than lysozyme in restricting the growth of fungal pathogens (Vandevenne et al., 2011). Based on the chitinolytic activity of lysozyme (Marquis, Montplaisir, Garzon, Strykowski, & Auger, 1982), Hall and co-authors postulated a synergistic action between CHIT1 and lysozyme, but could not find any evidence for this in antibacterial immunity in vitro (Hall et al., 2008). More recently, a synergistic action of both mammalian chitinases in antifungal immunity has been demonstrated in a rat model of aspergillosis (although this required disruption of the cell wall with caspofungin) (Verwer et al., 2013), a situation reminiscent of the concerted action of chitinases and ␤-1,3-glucanases in antifungal plant immunity (Jongedijk et al., 1995).
Natural mutations which disrupt functionality can give insights into the roles of human genes, as can the use of gene-disrupted animal models. Several dysfunctional mutations in CHIT1 have been found to be prevalent in human populations, without the association of any evident phenotype, suggesting that CHIT1 function is partially redundant (Boot et al., 1998). Indeed, a 24-bp duplication in Exon 10 of the chitotriosidase gene, causing the loss of the catalytic domain, is highly conserved in many human populations, but has not been found in primates, suggesting that it is a postspeciation event (Gianfrancesco & Musumeci, 2004). Specifically, this variant, also named H-allele, is almost absent in some West African (Burkina Faso: 0.2%)  and South African (South Africa: 0%) (Arndt, Hobbs, Sinclaire, & Lane, 2013) populations and showed the highest frequencies in Asiatic populations, suggesting it may have arisen after human migration out of Africa (Piras et al., 2007a,b).
Previous studies have hypothesized that the difference in duplication frequencies found between African populations in Benin, Burkina Faso and South Africa (Arndt et al., 2013;Malaguarnera et al., 2003) (98-100% homozygous wild-type) and those found in European populations, e.g. in Corsica and Sardinia (Piras et al., 2007a,b), Spain (Irún, Alfonso, Aznarez, Giraldo, & Pocovi, 2013), Portugal (Rodrigues, Sá Miranda, & Amaral, 2004) and the Netherlands (Boot et al., 1998) (<77% homozygous wild-type) may be due to the greater prevalence of parasitic infections in African populations, suggesting that chitotriosidase may possess an anti-parasitic function which has led to the maintenance of the wild-type allele in endemic areas. Overall, the frequency of the H-allele appears to vary significantly between populations (Arndt et al., 2013;Boot et al., 1998;Hise et al., 2003;Malaguarnera et al., 2003;Woo et al., 2014) and this variance in the frequency of functional chitotriosidase suggests that different populations vary in their need for the active protein. However, several studies could not find any correlation between rates of parasitic infection and duplication frequency in non-African areas endemic for parasitic infections (Hall et al., 2007;Hise et al., 2003).
We were therefore interested in studying CHIT1 genotype frequencies in a South American indigenous population with very low genetic admixture and very high exposure to chitin, through parasites and food, reflecting an ancestral lifestyle.

Ethical statement
Biological saliva specimens were taken safely and noninvasively, in full compliance with protocols approved by the Ethics Committee of the Università di Padova (2008). Informed consent was obtained from volunteers, or from their parents for underage volunteers. Project aims were presented to, and informed consent approved by, Awajún and Ashaninka indigenous organizations: OCCAAM (Central Organization of Awajún Communities of Alto Marañon) and ANAP (Pichis River Ashaninka Nationalities Association), respectively.

Peruvian Amerindians
In the Peruvian Andes and Amazons a high ethnic diversity is still preserved. Amerindians live in small communities of fifty up to several hundred people, and still maintain their original languages and bio-cultural adaptation to specific environmental conditions. Until the 1970s, most Amazonian communities of Peru were geographically isolated as they were cut off from the main routes of transportation, showing the highest prevalence of parasites and the lowest levels of water sanitation and national health-care of the country (Instituto Nacional de Salud, 2000;MINSA/OGE, 2002. Ethnic Amerindians involved in this study belong to five ethnic groups (  -Quechua-Cusco; NC: Huilloq (Cuzco Region), living in Andean highlands (altitude: 3000-4000 m above sea level); linguistic family: Quechua.
The populations are reciprocally isolated by both cultural (linguistic) and geographical barriers (see reciprocal distances in Table 1; mean: 1356 km), but, because of the small sample, the five Amerindian populations were considered as subpopulations and genetic data were finally clustered and discussed together, as 'Amerindian' population.

Assessment of exposure to chitin
As indicators of environmental exposure to chitin we used enteroparasitic prevalence and chitinous food consumption. The ingestion of chitin containing foods, such as crustaceans, insects and fungi, was assessed in the Amerindian and urban sample by standardized interviews and qualitative observations of ethnobiological habits.
Between December 2010 and March 2013, in Awajún, Ashaninka, Shipibo and Quechua-Cusco, the presence of intestinal parasites was assessed in a total sample population of 90 (from 14 to 34 volunteers for each NC: Awajún n = 34; Ashaninka n = 21; Shipibo n = 21; Quechua-Cusco n = 14) (5-12% of total villagers). Faecal samples were conserved in a 10% formol solution and analysed in the Helminthology Laboratory of Universidad Nacionàl de Trujillo, using standard protocols for direct copro-parasitological analysis (Instituto Nacional de Salud, 2003). Faecal samples were treated either by ether-sedimentation (Ritchie, 1948) or sugar-flotation (Sheather, 1923) preparation methods, and were examined with a binocular microscope (60-100× magnification), in duplicate, for detection and identification of eggs and cysts. For urbanized controls, epidemiological information was collected at local health institutions.

Genetic sampling
Genetic sampling was carried out with 85 ethnic Amerindian subjects and 50 urban controls. Saliva samples were collected with Oragene TM DNA Self-collection Kit (DNA Genotek, Canada) and DNA extracted as directed by the manufacturer. This device guarantees sample conservation at room temperature in hot and wet climatic conditions. The control population of 50 subjects was sampled in urban areas of Peru: Trujillo (n = 42) and Lima (n = 4) on the coast, and Tarapoto city (n = 4), in the Amazon. Most non-ethnic controls declared an Andean ancestry. Kinship relation between the volunteers was assessed by personal interviews and general pedigree and family trees were reconstructed, in the whole sample.
Unfortunately, volunteers involved in genetic and parasitological study rarely coincided in our study, because the two samplings were realized during different expeditions to the various remote communities; moreover, because of explicit requests by village leaders, parasitological analysis had to involve school-age children with suspected malnutrition while, contrarily, genetic sampling interested almost exclusively adult or elderly individuals.

Screening of the 24-bp duplication
For genetic screening of DNA samples for the 24 base pair duplication PCR was conducted in 15 l reactions consisting of 0.25 U of FastStartTaq (Roche), 2 mM MgCl 2 buffer, 0,2 l Forward Primer 5 -CCTGTCCAGAAGAGGTAGCC-3 , 0,2 l of reverse primer 5 -CCTCCAAATTCCACCACTG-3 , 200 M dNTPs, 1 l of genomic DNA, and 9 l of nuclease-free water. Primers were used at 250 nM final concentration. The Touchdown PCR program used was as follows: initial denaturation 94 • C for 4 min, followed by 10 cycles [94 • C for 40 s (denaturation) + 70 − 1 • C for 40 s (annealing) + 72 • C for 40 s (elongation)], 33 cycles [94 • C for 40 s, 60 • C for 40 s, 72 • C for 40 s], and a final extension at 72 • C for 7 min. Detection of 24-bp duplication on the PCR products was performed by DNA sequencing and/or by fragment separation by agarose gel electrophoresis in 4% standard molecular biology grade agarose gels (Sigma-Aldrich, UK) or on 3.5% MetaPhor TM Agarose (Lonza).
Amplicons were then purified with a Wizard ® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's protocol. Resultant elutes were then digested with 1 l HpaII (for G102S) or HinP1I (A442G/V) restriction enzyme (both from New England Biolabs) according to the manufacturer's instructions. Resultant digestions were then separated via gel electrophoresis run on 4% agarose (Sigma-Aldrich) or 3.5% MetaPhor TM Agarose (Lonza). The distinction between wild-type, heterozygous and homozygous mutant genotypes was made based on the size and Fig. 2. Example of high resolution gel electrophoresis screening for CHIT1 24 base pair duplication following PCR. Genomic DNA was amplified using primers described by (Boot et al., 1998) PCR products were separated on 3.5% high resolving agarose. Higher 99 bp bands indicate allele containing the 24 bp duplication, lower 75 bp bands indicate the wildtype allele (no duplication). A single higher band indicates homozygous mutant (HM), a single lower band indicates homozygous wild type (HW) and the presence of both bands indicates heterozygosity (Het).  Bierbaum et al. (2006) result in amplification of a 259 bp gene fragment from genomic DNA located in Exon 4 and the following intron of the CHIT1 gene. The forward primer introduces a mutation (T → C, denoted by an asterisk) located just 2 nucleotides upstream of the position of the SNP, introducing an Hpa II restriction site (C CGG, bold characters) for the wildtype G102 SNP (resulting in a 240 bp and a 19 bp fragment after restriction) but not the mutant S102 SNP (uncleaved 259 bp fragment). (B) Example of gel electrophoresis screening for CHIT1 G102S SNP following PCR and digestion with Hpa II. Higher bands indicate undigested allele containing S102 SNP, lower bands indicate large fragment of digested wild G102 allele. Single high band indicates homozygous mutant (HM), single lower band indicates homozygous wild type (HW) and presence of both bands indicates heterozygosity (Het).

Data analysis
Genotypic frequencies were compared with frequencies expected by Hardy-Weinberg equilibrium (HWE). Based on general pedigree data, inbreeding coefficients and kinship were calculated by KinInbcoef program (Bourgain, 2005), and allelic frequency values were corrected by kinship degree.

Exposure to chitin: Diet and enteroparasites
Ethnobiological information confirmed traditional dietary habits including daily consumption of insects, crustaceans and mushrooms, and a very rare presence of pets, in the Amerindian sample, especially in Awajún, Ashaninka, Quechua-Lamas and Shipibo (before migrating to urban Lima). In contrast, Quechua-Cusco people reported only rarely consuming insects, crustaceans and mushrooms. Similarly, urban controls declared to eat, weekly, crustaceans, and, very rarely, mushrooms.
In contrast, lower parasite prevalence of mainly protozoan parasites characterizes the control population of urbanized Peruvians.

Chitotriosidase deficiency
The 24-bp duplication causing catalytic deficiency was highly prevalent (Table 3), with a frequency of 47.06% (Kinship correction: 45.99%) in the whole indigenous population, and 27.55% (Kinship correction: 28.88%) in non-indigenous controls. Genotypic frequencies were consistent with the Hardy-Weinberg Equilibrium (HWE), within each indigenous population, in total Amerindians, and controls.
Next, we screened a subset (unselected) of 55 indigenous Peruvians (33 Amazonians and 22 Quechua) and 28 Peruvian nonindigenous controls for previously described non-synonymous SNPs in the CHIT1 gene, G102S and A442G (Bierbaum et al., 2006) as well as A442V (Lee, Waalen, Crain, Smargon, & Beutler, 2007). Parasite prevalence is calculated as frequency of infected individuals for each community sampled. Prevalence is given by species (or by phyla) in the four communities studied. * Parasites thought to be chitin-bearing are marked with asterisk. The method used does not discriminate A442G from A442V; hence results are given collectively as A442G/V for the latter SNP (Table 4). Similarly to the results obtained for the H-allele, the genotype screening for the G102S SNP showed a high frequency for the mutation, while the frequency of A442G/V was somewhat lower. There was no significant departure from HWE in any population.

Discussion
Both ethnobiological and parasitological studies gave evidence of a conserved traditional lifestyle, strongly influenced by environmental factors and, specifically, by chitin-containing parasites and traditional local food including chitin-bearing invertebrates and mushrooms. The absence of Taenia and Fasciola is most likely related to the very low presence of livestock in indigenous Amazonian areas, where 'original' ethno-ecological features regarding nutrition and lifestyle are still predominant. Overall, the absence of such zoonoses, together with a high prevalence of soiltransmitted helminths (nematodes) can be considered an ancestral parasitological condition, as confirmed by pre-Columbian fossil remains (Araujo, Reinhard, Ferreira, & Gardner, 2008;Sianto et al., 2009). Furthermore, the very low prevalence of filarial nematodes   , South Africa (100%) (Arndt et al., 2013), Spain (Basques) (88%) (Piras et al., 2007a,b), The Netherlands (77%) (Boot et al., 1998), Sicily (73%) (Piras et al., 2007a,b), India (60%) (Choi et al., 2001), Papua New Guinea (88%) (Hise et al., 2003), Chinese Han, Taiwan (42%) (Chien, Chen, & Hwu, 2005), Mexico (76%) (Juárez-Rendón, Lara-Aguilar, & García-Ortiz, 2013), Brazil (74%) (Adelino et al., 2013) and Amerindians of Peru (52%). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) in the Amazon is a rare feature for worldwide tropical areas, confirming a broadly 'better' parasitological condition/reduced parasitological burden, in original Amerindians. Although no correlation analysis between CHIT1 genotype and parasite infection is possible with present data, our key finding is that the prevalence of chitotriosidase deficiency in the indigenous sample is very high, with up to 26% of individuals having no functional chitotriosidase. Thus, we can conclude that chitin-containing enteroparasites are not exerting any significant selective pressure for functional chitotriosidase in our sample. The prevalence of chitotriosidase deficiency is lower in non-indigenous controls, which is likely to result from genetic admixture with Europeans and Africans in recent times.
The genotype screening for the G102S SNP similarly shows a high frequency for the mutation (allelic freq. = 36-52%), though in this case allele frequencies were similar in indigenous and non-indigenous samples, but higher compared to other human populations (allelic freq. = 27-37%) (data available from the literature (Lee et al., 2007), and from ENSEMBL genomic database: http://www.ensembl.org/index.html; ref seq.: rs2297950). For the A442G/V SNPs, the mutant allele frequency (16-32%) is lower compared to the G102S SNP, but still higher compared to the frequencies (allelic freq. = 7-17%) reported for other populations (ENSEMBL, ref. seq.: rs1065761). Previous studies have indicated that the effect of the G102S SNP on enzymatic activity varies from undetectable to significant, depending on the synthetic assay substrate used (Bussink et al., 2009). Similarly, it has been determined that the A442V SNP decreases the enzymatic activity of expressed chitotriosidase (Lee et al., 2007), while the A442G SNP may be associated with increased risk of atopic disorders in childhood (Kim et al., 2013).
No significant deviation from expected genotypic frequencies under HWE were found for any of the three studied polymorphisms, as also found in several other human populations (Piras et al., 2007a,b). This suggests that there is no strong selective pressure against the homozygous mutated phenotypes. In previous work, we proposed that human expansion to temperate regions, and the gradual improvement of hygienic conditions, may have reduced exposure to chitin from parasites and diet enabling the conservation of dysfunctional mutations in the CHIT1 gene (Cozzarini et al., 2009;Paoletti, Norberto, Damini, & Musumeci, 2007). However, the current data from Peruvian Amerindians with high enteroparasite prevalence do not support this hypothesis. This finding is in line with our previous inability to find a correlation between CHIT1 genotype and susceptibility to hookworm infection (Hall et al., 2007) (Fig. 5).
Considering that the first inhabitants of the New World descended from eastern Asian populations between 6 and 30 thousand years ago, it is plausible to assume that the high frequencies of the mutation (>40%) result from a founder effect in Amerindians. Both linguistic and genetic data suggest that the populations considered in our study are reciprocally isolated: first, Awajún, Ashaninka, Shipibo and Quechua belong to four different linguistic macro-families, being separated at higher levels of classification (Cavalli-Sforza, Piazza, Menozzi, & Mountain, 1988); second, population genetic information based on different genetic markers in South-Amerindians confirm significant genetic divergence between the ethno-linguistic groups considered in this study (Bisso-Machado, Bortolini, & Salzano, 2012). Thus, our relatively small sample of 'Peruvian Amerindians' is more broadly representative of 'South American Amerindians'.
Chitotriosidase has been suggested to have a role in resistance to malaria, as the H-allele is almost absent in highly malarious areas of Africa. An anti-malarial role of the 24-bp duplication has been hypothesized for European and Mediterranean populations living in regions endemic for malaria until recent times (Piras et al., 2007a,b). The interaction mechanism proposed by Di Luca and colleagues (Di Luca et al., 2007), concerns the fact that Plasmodium's chitinase is required for mosquito's peritrophic membrane digestion. Human CHIT1, which is increased in blood during acute infection (Barone, Simporé, Malaguarnera, Pignatelli, & Musumeci, 2003), possibly contributes to this process (Di Luca et al., 2007). Therefore, the catalytically inactive form of human CHIT1 has been suggested to reduce malaria transmission at subsequent bites, thus high frequencies of the duplication may confer some level of protection against malaria at the community level. Indeed, a positive association between H-allele frequency and altimetric distribution of malaria was found in Sardinian and Sicilian cohorts (Piras et al., 2007b).
However, our previous study in Papua New Guinea failed to find a correlation between CHIT1 genotype and malarial status in a P. falciparum mesoendemic region (Hall et al., 2007). As noted by previous authors, the inactivated, transmission-reducing CHIT1 may not be required in Benin and Burkina Faso populations, which display high frequencies for genetic conditions conferring natural resistance against malarial pathogenicity, such as thalassemia and G6PDH deficiency , as well as Duffy factor negativity (Gething et al., 2012), that may encourage selection for 'anti-parasitic' (enzymatically active) CHIT1 rather than 'antimalarial' (enzymatically inactive) CHIT1. However, the duplication was present in a Papua New Guinea population with high malaria and a high frequency of deleterious malaria resistance genes (Hall et al., 2007).
Historical and epidemiological data suggest Plasmodium species spread in the New World after the European conquest (De Castro & Singer, 2005), and malaria became mesoendemic in the Peruvian Amazon lowlands at the beginning of the 17th century. Moreover, malaria vector distribution in Peru is strictly limited by altitude and Quechua populations living in the highlands (>2500 m above sea level) thus are not exposed to this pathogen. In our study, an effect of altitude could not be confirmed as the duplication resulted conserved in Quechua-Cusco and Quechua-Lamas, similarly to Amazonians. However, known rates of malarial infection in Peru are low in comparison to West African population  and other South American countries, even amongst nonindigenous populations (Gething et al., 2012), therefore it is not excluded that the 24-bp duplication, conserved as founder effect amongst Amerindian populations, may secondarily control malaria transmission, and high frequencies of the mutant allele may contribute in controlling the spread of malaria in the Amazon.
Finally, it must be considered that a significant prevalence of the two SNPs considered, together with other variants causing chitotriosidase deficiency (SNP G354R; deletion: E/I-10 delGAgt) (Grace, Balwani, Nazarenko, Prakash-Cheng, & Desnick, 2007) has been described in Black South Africans (Arndt et al., 2013), where the 24-bp duplication is absent. Thus, these authors inferred that the loss of chitinolytic function has arisen in modern humans by different genetic events.

Conclusions
Many scientists investigating bio-anthropological issues in isolated ethnic populations have stressed the need for studying specific epidemiological and immunological features to adopt appropriate health-care interventions (i.e. Hurtado et al., 2005, Hurtado, Frey, Hurtado, Hill, & Baker, 2008. Our evidence shows that in Amazonian Amerindians, who have been living relatively isolated until only a few decades ago, CHIT1 genotypes are similar to those found in Asian populations, in marked contrast to African and European populations. The high frequency of the dysfunctional CHIT1 genotypes is consistent with a founder effect and does not corroborate previous suggestions of a persisting anti-parasitic function of this gene. However, demographic and environmental conditions in the Amazon are rapidly changing, and new epidemic burdens are arising (e.g. filariasis in Brazilian Amazon or zoonotic infections from the introduction of cattle in indigenous areas), therefore new immunoparasitological challenges may impact on Amazonian populations with high prevalence of chitinase deficiency, in the near future.
Chitotriosidase expression and activity have been suggested as efficient markers of immune-mediated disorders as Gaucher's, Atherosclerosis or Alzheimer's Grace et al., 2007), but the high prevalence of chitotriosidase deficiency in our South Amerindian sample precludes this diagnostic application in South American subjects with marked ethnic ancestry.