Analysis of heat shock protein 70 gene polymorphisms Mexican patients with idiopathic pulmonary fibrosis

Hsp70s, together with their Hsp40 co-chaperones, are the most prominent chaperone
families that participate in chaperone-assisted proteosomal degradation of misfolded
proteins 31]. Previously published functional studies have highlighted the importance of HSP70
to attenuate the abnormal lung remodelling after injury in experimental models 32], 33]. Accordingly, it has been shown that upregulation of HSP70 significantly decreases
the inflammatory and fibrotic response in bleomycin-induced pulmonary damage, blocking
the production of TGF-?1 33]. Likewise, there is evidence indicating that gefitinib-induced exacerbation of bleomycin-induced
lung fibrosis is mediated by suppression of pulmonary expression of HSP70 34]. However, studies in human IPF are scarce. Intriguingly, it was found that a subgroup
of IPF patients has significantly greater extent of anti-HSP70 humoral and cellular
autoreactivities compared with healthy controls. Moreover, abnormal anti-HSP70 humoral
autoimmunity was associated to poor outcome. Whether the development of this autoreactivity
is related to some HSP70 polymorphisms is unknown 35]. Interestingly, the autoimmune-associated HLA-B8-DR3 haplotypes seems to include
the HSPA1B 1267A/G polymorphism 36]. Likewise, it has been reported in the Chinese population that the A allele is more
predominant in patients with enterocutaneous fistulas than in healthy controls 37]. In this study, we determined variants of HSP70 genes in IPF patients and healthy controls from Mexican ancestry. The most striking
findings confirmed by a multivariate analysis were that the GA and AA genotypes of the HSPA1B (rs1061581) polymorphism were associated with a lower risk of IPF. Also, AG genotype
and the AG?+?GG genotypes (in a dominant model) of the SNP HSPA1L (rs2227956) were also associated with a lower risk to develop IPF. On the other hand,
the HSPA1L (rs2075800) TT genotype was significantly associated with susceptibility to IPF.

The polymorphism rs2227956 is located in the coding region of the HSPA1L gene and leads to an amino acid change at position 493 from a non-polar hydrophobic
Met to a polar neutral Thr. Amino acid 493 is present in the 18 kDa peptide-binding
domain on the beta sheet that forms the floor of the peptide binding groove 38]. The HSPA1L polymorphic G allele translates to a Met residue, an hydrophilic amino acid that
may affect the interaction of the HSP70 with hydrophobic proteins and consequently
impairs its ability to assemble and transport proteins within cells 38], 39]. Intriguingly, a recent meta-analysis study indicated that individuals with HSPA1B AG/GG genotype, which seem to protect from IPF, have an increased risk of cancer
40]. Previous studies have highlighted the functional consequences of different HSP-70
gene polymorphisms. The polymorphisms +190 C of HSPA1A, HSPA1B-179 CT and HSPA1B 1267 AG have been linked with significant alterations in the mRNA and protein expression
25], 26]. Also an important functional effect at protein level of the polymorphism HSP70-HOM +2437 AG (Met493Thr), affecting the substrate specificity and chaperone activity
of HSP-70 HOM, has been described 12], 27]. The possible mechanisms of HSP70 gene-disease associations in IPF are unclear. In this context, strong evidence has
revealed the accumulation of unfolded and misfolded proteins with severe endoplasmic
reticulum (ER) stress in alveolar epithelial cells lining areas of fibrosis 41]. It is possible that the functional effect of the HSPA1B 1267 AG polymorphism in the differential expression of HSPA1B mRNA 26] might influence the protein expression of pro-fibrotic proteins associated to the
pathogenesis of IPF. Moreover, it has been shown that the same cells also display
activation of pro-apoptotic pathways 42]. In addition, ER stress may contribute to the pathogenesis of IPF through the induction
of epithelial to mesenchymal transition, which may play a role in the expansion of
the fibroblast population. Consequently, a transient increase in the expression of
heat shock proteins is critical to prevent alveolar epithelial cell death and the
shift of epithelial cells to a mesenchymal phenotype. In this perspective, an age-dependent
decrease in the ability of different cell types to synthesize HSP70 has been observed,
and moreover, some gene variants seem to contribute to this decrease 43].

In our study, the combination of the two genotypes associated with protection was
only found in healthy controls and not in IPF patients (p?=?0.005). In comparison with former data of associated functional polymorphisms 12], 18], 26], 27], 44], it seems that there is no direct relationship among them and those reported herein,
since these previous studies show that HSP70 protects from fibrosis development. Strikingly,
our results point out that genotypes involved in the decreased induction of these
proteins might provide protection against IPF. Moreover, the association between these
polymorphisms and IPF protection may be due to LD with other adjacent genes. In this
regard, an LD has been reported between the TNF locus and HSPA1B45].

To determine whether the associations described in the three models of inheritance
are independent of LD between the SNPs analyzed, a multivariate analysis was performed.
As a result we confirmed that these associations are independent of LD. The differences
in the association with IPF between the three models of inheritance may indicate that
the segregation of one or two genotypes of protection or risk act independently, either
as protective or risk IPF factors, respectively. The present study has some limitations
and replications in other populations are needed to verify these findings, since polymorphisms
suggesting fibrosis protection are relatively rare. Notwithstanding, we were able
to confirm HSP70 polymorphisms association with the protection to IPF with a statistical power greater
that 80 %, even with the stringently defined number of IPF cases studied. Another
limitation was the imbalanced distribution of males and females among IPF and control
group. In this regard, a possible bias of the genetic association analysis may be
due to the gender disparities. However, in previous genetic-association studies of
MHC genes, no marked differences in the MHC genes frequencies have been detected between
males and females from Mexican ancestry 6]. Furthermore, it is well know that IPF is more frequent in Mexican males and due
to the age of onset of the disease, it is more frequent to have healthy females than
males.

Finally, we compared the frequencies of HSP70 SNPs genotypes between Mexicans and other ethnic groups (Additional file 1: Table S1). In this analysis we found that the homozygous TT genotype of the polymorphism
rs2075800 of the HSPA1L gene is the most common genotype, whereas CC genotype is the least frequent genotype
in Italian Caucasians 46] and Croatian 47] subjects. We also observed that in Asian populations including Chinese 48] and Taiwanese 49] as well as in Mexicans, the CC genotype is frequent and the genotype TT is uncommon.
The genotypic frequencies HSPA1L with rs2227956 are similar in all population groups. Likewise, for the HSPA1A rs1043618 gene, the GG genotype is more frequent in Chinese, Taiwanese, Polish 50], 51] and Mexicans; in contrast, the most common in Italians is heterozygous CG, and the
least frequent in all populations, is the homozygous CC.