Mammalian NPC1 genes may undergo positive selection and human polymorphisms associate with type 2 diabetes

The NPC1 gene encodes a large multi-domain protein involved in the intracellular trafficking of sterols. Mutations in the gene are responsible for a rare and fatal lipid storage disorder, Niemann-Pick disease type C. The product of NPC1 resides in the limiting membrane of late endosomes and lysosomes where it facilitates lipid transport to various cellular compartments (reviewed in [1]). The protein displays 13 transmembrane domains, and three large loops are present in the lumen of the endosome (Figure 1) [2]. Interaction with lipid substrates is mediated by the most N-terminal luminal loop (loop 1) and by a sterol-sensing domain (SSD) which comprises five central transmembrane regions [2] (Figure 1). Recent works showed that the subcellular localization of NPC1 has been exploited by viruses of the Filoviridae family for host invasion [3–5]. Thus, viruses such as Ebola and Marburg require NPC1 protein expression for productive infection and the second luminal domain of NPC1 binds directly and specifically to the GP1 viral glycoprotein [3]. Consistently, primary fibroblasts from human Niemann-Pick type C1 disease patients are resistant to infection by filoviruses [4].

Mice lacking Npc1 function display a phenotype recapitulating Niemann-Pick disease type C [6], whereas haploinsufficiency for the gene results in weight gain and insulin resistance [7, 8]. In fact, Npc1 ^+/- mice display increased adiposity and adipocyte hypertrophy; these animals also show dyslipidemia and higher plasma glucose levels compared to their wild-type litter mates. In line with this evidence, a nonsynonymous polymorphism (rs1805081, His215Arg) in the human NPC1 gene has recently been associated with severe and early onset obesity in European populations [9]. A subsequent study confirmed the predisposing role of rs1805081 to obesity and increased body mass index (BMI) in Europeans, but found no association between the variant and type 2 diabetes (T2D) or fasting plasma lipid levels [10]. Conversely, the effect on obesity risk and higher BMI of the NPC1 SNP in Asian populations is still controversial [11, 12]. The molecular mechanisms underlying the association between genetic variation in NPC1 and metabolic phenotypes remain to be clarified. However, analysis of Npc1 mutant mice revealed that these animals are characterized by increased liver accumulation of triacylglycerol [7], higher hepatic expression of caveolin-1 [13], a protein involved in liver lipid metabolism [14], and of sterol regulatory element-binding proteins (SREBPs) [15]. These observations suggest that mutations or polymorphisms in NPC1 result in alteration of hepatic lipid homeostasis eventually leading to weight gain and insulin resistance.

Adaptations to diet and to pathogen exposure are thought to have represented a powerful driving force throughout the evolutionary history of mammals [16]. Thus, we performed a phylogenetic analysis of NPC1 genes in mammals and a population genetics study of diversity in human populations. We identified three residues that have been targets of positive selection, possibly mediated by filovirus-exerted selective pressure. No selection signature was detected in present-day human populations, but analysis of nonsynonymous polymorphisms identified a variant (Ile642Met) in the SSD domain that affects a highly conserved position. This variant and NPC1 haplotypes were found to modulate the risk of T2D (but not BMI or obesity) in a population from Saudi Arabia.

During mammalian evolution genes involved in diet and immune response have been preferential targets of positive selection [16], highlighting the role of nutrient availability/preferences and pathogens as powerful selective forces. The protein product of NPC1 plays a central role in lipid metabolism, as it acts as a cholesterol transporter and its transcription is regulated by the SREBP pathway [1]. Conversely, the gene does not participate in immune response, but is exploited by members of the filovirus family as an intracellular receptor that mediates the late steps of viral invasion [3–5]. Evidence has indicated that genes directly involved in antiviral response or acting as viral receptors (for example, HAVCR1, CD4) display domains evolving under positive selection as the result of a genetic conflict with extant or extinct viral species [38–46]. Positive selection at these host genes may result from adaptation either to increase viral recognition and restriction efficiency or to avoid binding of specific viral components. Our evolutionary analysis in mammals indicated a predominant role of purifying selection in driving the evolution of NPC1 but also identified few positions that have been targeted by positive selection. Specifically, maximum-likelihood ratio tests indicated that three residues in the N-terminal portion of luminal loop 2 evolved under positive selection; these codons are located in close proximity to each other, and selection was confirmed by an independent REL analysis. PAML also identified one positively selected site in luminal loop 1, but this was not supported by REL, suggesting that it may represent a false positive, as the M8 model has been shown to be more prone than REL to false positive results when a relatively high number of sequences (species) is used for analysis [47]. These results suggest that the selective pressure responsible for positive selection in NPC1 stems from pathogens rather than from dietary changes. Indeed, a recent study has indicated that luminal loop 2 is necessary and sufficient to bind filovirus GP1 protein directly and to mediate productive infection [3]; the authors were able to map the GP1 residues involved in engaging loop 2 and determined that they are conserved among filoviruses [3]. This observation, together with evidence showing that NPC1 is required for infection of both human and rodent cells by distantly related viral species, strongly suggests that the cholesterol transporter is a necessary factor for most members of the Filoviridae family [3–5]. These pathogens display a wide host range in mammals [48] and are thought to have affected vertebrates for millions of years, as testified by the detection of filovirus-derived elements in the genome of both eutherians and marsupials [49]. Thus, we suggest that the positively selected sites we identified in luminal loop 2 evolved in response to a host-filovirus arms race and might represent relevant residues in mediating GP1 binding.

Population genetic analysis of NPC1 in humans revealed no evident signature of natural selection in loop 2 or any other gene region, although we cannot exclude that weak or geographically-restricted selective events have acted on the gene. With respect to filovirus infection, this might not be surprising as the known human pathogens Ebola and Marburg viruses are highly virulent agents that rapidly kill infected individuals, a feature that possibly limits their spreading in human populations [50] and makes them unlikely candidates to play a role as selective agents. Genetic diversity in human NPC1 has nevertheless been recently associated with metabolic dysfunction, this association being based on the central role of the gene in lipid trafficking. Specifically, the His215Arg (rs1805081) variant in luminal loop 1, which is involved in cholesterol binding, was shown to associate with obesity in populations of European descent [9, 10]. It has been proposed that alleles responsible for obesity and T2D might have evolved as 'thrifty' variants in ancient populations [51, 52]. In line with this hypothesis, selection signatures have been detected for a few polymorphisms associated with these conditions [53, 54], although this does not seem to be the case for NPC1. Nonetheless, inspection of nonsynonymous SNPs located in the gene revealed that, in addition to the above mentioned variant in loop 1, a polymorphism (Ile642Met, rs1788799) in the SSD domain segregates at relatively high frequency in human populations and affects an isoleucine residue which is conserved in all the mammals we analyzed.

We thus reasoned that this SNP might affect NPC1 function and modulate metabolic phenotypes. We tested this hypothesis in a large cohort of subjects from Saudi Arabia, a region where the prevalence of obesity and T2D is very high [55–57]. The previously described association between rs1805081 and obesity [9, 10] was not replicated in the Saudi sample, although the relatively lower minor allele frequency (MAF) of the variant in this population (12%) compared to Europeans (ranging from 25% to 40%) might have limited our detection power. No effect on BMI or obesity was detected in the Saudi cohort for the Ile642Met variant either. Similarly, the role of the His215Arg variant in predisposing to obesity was not observed in a cohort of Chinese children [12], although a possible interaction between this (and other) variant and sedentary behavior has been described in a population of the same ethnicity [11]. Recently, a meta-analysis of rs1805081 on obesity risk in Europeans also revealed a weak effect of the polymorphism on body fat percentage, but not on BMI or on the odds of being obese [58]. One possibility to explain these contrasting results is that variants in NPC1 interact with environmental cues, as suggested by the Chinese study [11] and possibly with additional genetic factors. This seems to be the case for Npc1 ^+/- mice: these animals develop increased adiposity and metabolic disturbances but the phenotype depends on both fat intake and genetic background [7, 59]. These animals also present with increased fasting plasma glucose levels, glucose intolerance, and insulin resistance, indicating a T2D phenotype [7, 59]. Somehow in contrast with these results, a recent study indicated that heterozygosity for a hypomorphic Npc1 mutation on the C57BL/6J 'metabolic syndrome' genetic background protects old male mice, but not females, from weight gain [60]. Overall, these observations suggest that Npc1 genetic variation interacts with diet, sex and with one or more gene(s) in modulating metabolic phenotypes.

A possible association between the two NPC1 variants and T2D was analyzed in the Saudi cohort. Overweight and obesity are strong risk factors for the development of T2D; genetic susceptibility is nevertheless believed to play a stronger role in non-obesity related T2D [61]. Thus, we verified the effect of rs1805081 and rs1788799 on diabetes susceptibility by taking BMI into account; a significant association was detected between rs1788799 and T2D, with a predisposing role for the derived 642Met allele.

Several metabolic traits are sexually dimorphic in humans and/or show sex-specific heritability linked to the autosomes [62]. Thus, it was suggested that variants with a sex-specific effect might be difficult to detect without separating the sexes or modeling for gender-based differences [62]. Testing for interaction with sex in our cohort indicated the presence of a significant effect; stratification of the population on the basis of gender revealed that the association is driven by male subjects. This was even more evident when haplotype analysis using the two coding variants was performed. Notably, two major haplotypes showed an opposite effect on T2D susceptibility in men only, and the effect was evident in both obese and non-obese individuals. An interaction between gender and genetic factors has been described for some other genes involved in T2D [63–66]; the reasons underlying these sex-specific events remain to be elucidated and might include a role for sex hormones, epistatic effects with X-linked variants, or differences in dietary habits and lifestyle between the sexes that, in turn, interact with the genetic status.

Further analyses on plasma lipid levels showed the presence of different associations with NPC1 haplotypes in men and women. Nonetheless, these effects were generally weak and should be interpreted with caution. The stronger effect was detected for triglyceride levels. Thus, in men a minor haplotype unrelated to T2D susceptibility was found to associate with higher levels, whereas in women the two major haplotypes that predispose or protect men from diabetes were found to be associated with higher and lower triglyceride levels, respectively.

Data reported here indicate that NPC1 has evolved adaptively in mammals and that the underlying selective pressure might be virus-driven. No selection signature was detected in present-day human populations, but analysis of nonsynonymous polymorphisms showed that a variant (Ile642Met) in the SSD domain affects a highly conserved position. This variant and haplotypes comprising Ile642Met and the previously described His215Arg polymorphism were found to modulate the risk of T2D in a population from Saudi Arabia with a sex-specific effect. Analysis of additional cohorts will be instrumental for clarifying the role of the two NPC1 variants on plasma lipid levels and T2D susceptibility. Our results indicate that haplotype analysis (as opposed to single variant association) and modeling for sex-specific effects are strongly recommended when NPC1 genetic variability is analyzed.