Presence of three mutations in the fumarylacetoacetate hydrolase gene in a patient with atypical symptoms of hereditary tyrosinemia type I.

Hereditary tyrosinemia type 1 (HT1), the most severe disease of the tyrosine catabolic pathway, is caused by a deficiency of fumarylacetoacetate hydrolase (FAH). More than 90 disease-causing variants have been identified in the fah gene. We investigated the molecular defect in a patient who presented atypical symptoms for the disease. No immunoreactive FAH was found in the liver and RNA analysis by RT-PCR suggested the presence of splicing mutations. Indeed, the patient was revealed to be a compound heterozygote for IVS6-1 g- > t and two new variants, namely p.V259L and p.G398E. Using splicing minigene constructs transfected in HeLa cells, the c.775G > C variant (p.V259L) was shown to affect partially exon 9 splicing thereby allowing the production of some full-length double-mutant FAH transcripts. The p.G398E variant had a major impact on enzyme activity, which was worsened by the p.V259L variant. Surprisingly, the double mutant protein was expressed to similar level as the wild-type protein upon transfection in HeLa cells but was absent in the patient liver extract, suggesting a higher propensity to be degraded in the hepatocellular context.

Small heat shock proteins: multifaceted proteins with important implications for life.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.

Identification of proteins interacting with the mitochondrial small heat shock protein Hsp22 of Drosophila melanogaster: Implication in mitochondrial homeostasis.

The small heat shock protein (sHsp) Hsp22 from Drosophila melanogaster (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient in vitro chaperone, its function within mitochondria in vivo remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.

Biochemical and Clinical Aspects of Hereditary Tyrosinemia Type 1.

Inborn errors of metabolism (IEMs) are a group of diseases involving a genetic defect that alters a metabolic pathway and that presents usually during infancy. The tyrosine degradation pathway contains five enzymes, four of which being associated with IEMs. The most severe metabolic disorder associated with this catabolic pathway is hereditary tyrosinemia type 1 (HT1; OMIM 276700). HT1 is an autosomal recessive disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. Although a rare disease worldwide, HT1 shows higher incidence in certain populations due to founder effects. The acute form of the disease is characterized by an early onset and severe liver failure while the chronic form appears later and also involves renal dysfunctions. Until 1992 the only treatment for this disease was liver transplantation. Since then, NTBC/Nitisone (a drug blocking the pathway upstream of FAH) is successfully used in combination with a diet low in tyrosine and phenylalanine, but patients are still at risk of developing hepatocellular carcinoma. This chapter summarizes the biochemical and clinical features of HT1.

Molecular Aspects of the FAH Mutations Involved in HT1 Disease.

Hereditary tyrosinemia type 1 (HT1) is caused by the lack of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. Up to now, around 100 mutations in the FAH gene have been associated with HT1, and despite many efforts, no clear correlation between genotype and clinical phenotype has been reported. At first, it seems that any mutation in the gene results in HT1. However, placing these mutations in their molecular context allows a better understanding of their possible effects. This chapter presents a closer look at the FAH gene and its corresponding protein in addition to provide a complete record of all the reported mutations causing HT1.

Molecular Pathogenesis of Liver Injury in Hereditary Tyrosinemia 1.

Untreated HT1 rapidly degenerates into very severe liver complications often resulting in liver cancer. The molecular basis of the pathogenic process in HT1 is still unclear. The murine model of FAH-deficiency is a suitable animal model, which represents all phenotypic and biochemical manifestations of the human disease on an accelerated time scale. After removal of the drug 2-(2-N-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), numerous signaling pathways involved in cell proliferation, differentiation and cancer are rapidly deregulated in FAH deficient mice. Among these, the Endoplasmic reticulum (ER) pathway, the heat stress response (HSR), the Nrf2, MEK and ERK pathways, are highly represented. The p21 and mTOR pathways critical regulators of proliferation and tumorigenesis have also been found to be dysregulated. The changes in these pathways are described and related to the development of liver cancer.

The growing world of small heat shock proteins: from structure to functions.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

Identification of circulating microRNAs during the liver neoplastic process in a murine model of hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe inborn error of metabolism, impacting the tyrosine catabolic pathway with a high incidence of hepatocellular carcinoma (HCC). Using a HT1 murine model, we investigated the changes in profiles of circulating and hepatic miRNAs. The aim was to determine if plasma miRNAs could be used as non-invasive markers of liver damage in HT1 progression. Plasma and liver miRNAome was determined by deep sequencing after HT1 phenotype was induced. Sequencing analysis revealed deregulation of several miRNAs including let-7/miR-98 family, miR-21 and miR-148a, during manifestation of liver pathology. Three miRNAs (miR-98, miR-200b, miR-409) presenting the highest plasmatic variations among miRNAs found in both plasma and liver and with >1000 reads in at least one plasma sample, were further validated by RT-qPCR. Two of these miRNAs have protein targets involved in HT1 and significant changes in their circulating levels are detectable prior an increase in protein expression of alpha-fetoprotein, the current biomarker for HCC diagnosis. Future assessment of these miRNAs in HT1 patients and their association with liver neoplastic lesions might designate these molecules as potential biomarkers for monitoring HT1 damage progression, improving diagnosis for early HCC detection and the design of novel therapeutic targets.

Molecular changes associated with chronic liver damage and neoplastic lesions in a murine model of hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is the most severe inherited metabolic disease of the tyrosine catabolic pathway, with a progressive hepatic and renal injury and a fatal outcome if untreated. Toxic metabolites accumulating in HT1 have been shown to elicit endoplasmic reticulum (ER) stress response, and to induce chromosomal instability, cell cycle arrest and apoptosis perturbation. Although many studies have concentrated on elucidating these events, the molecular pathways responsible for development of hepatocellular carcinoma (HCC) still remain unclear. In this study the fah knockout murine model (fah(-/-)) was used to investigate the cellular signaling implicated in the pathogenesis of HT1. Fah(-/-) mice were subjected to drug therapy discontinuation (Nitisinone withdrawal), and livers were analyzed at different stages of the disease. Monitoring of mice revealed an increasing degeneration of the overall physiological conditions following drug withdrawal. Histological analysis unveiled diffuse hepatocellular damage, steatosis, oval-like cells proliferation and development of liver cell adenomas. Immunoblotting results revealed a progressive and chronic activation of stress pathways related to cell survival and proliferation, including several stress regulators such as Nrf2, eIF2α, CHOP, HO-1, and some members of the MAPK signaling cascade. Impairment of stress defensive mechanisms was also shown by microarray analysis in fah(-/-) mice following prolonged therapy interruption. These results suggest that a sustained activation of stress pathways in the chronic HT1 progression might play a central role in exacerbating liver degeneration.

Annexin A2 is SUMOylated on its N-terminal domain: regulation by insulin.

Insulin receptor (IR) endocytosis requires a remodelling of the actin cytoskeleton. We show here that ANXA2 is SUMOylated at the K10 located in a non-consensus SUMOylation motif in the N-terminal domain. The Y24F mutation decreased the SUMOylation signal, whereas insulin stimulation increased ANXA2 SUMOylation. A survey of protein SUMOylation in hepatic Golgi/endosome (G/E) fractions after insulin injections revealed the presence of a SUMOylation pattern and confirmed the SUMOylation of ANXA2. The construction of an IR/ANXA2/SUMO network (IRASGEN) in the G/E context reveals the presence of interacting nodes whereby SUMO1 connects ANXA2 to actin and microtubule-mediated changes in membrane topology. Heritable variants associated with type 2 diabetes represent 41% of the IRASGEN thus pointing out the physio-pathological importance of this subnetwork.

Heat shock response associated with hepatocarcinogenesis in a murine model of hereditary tyrosinemia type I.

Hereditary Tyrosinemia type 1 (HT1) is a metabolic liver disease caused by genetic defects of fumarylacetoacetate hydrolase (FAH), an enzyme necessary to complete the breakdown of tyrosine. The severe hepatic dysfunction caused by the lack of this enzyme is prevented by the therapeutic use of NTBC (2-[2-nitro-4-(trifluoromethyl)benzoyl] cyclohexane-1,3-dione). However despite the treatment, chronic hepatopathy and development of hepatocellular carcinoma (HCC) are still observed in some HT1 patients. Growing evidence show the important role of heat shock proteins (HSPs) in many cellular processes and their involvement in pathological diseases including cancer. Their survival-promoting effect by modulation of the apoptotic machinery is often correlated with poor prognosis and resistance to therapy in a number of cancers. Here, we sought to gain insight into the pathophysiological mechanisms associated with liver dysfunction and tumor development in a murine model of HT1. Differential gene expression patterns in livers of mice under HT1 stress, induced by drug retrieval, have shown deregulation of stress and cell death resistance genes. Among them, genes coding for HSPB and HSPA members, and for anti-apoptotic BCL-2 related mitochondrial proteins were associated with the hepatocarcinogenetic process. Our data highlight the variation of stress pathways related to HT1 hepatocarcinogenesis suggesting the role of HSPs in rendering tyrosinemia-affected liver susceptible to the development of HCC.

Annexin A1 is regulated by domains cross-talk through post-translational phosphorylation and SUMOYlation.

Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca(2+) and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca(2+)-free conformation implying that Ca(2+) binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications.

Protection from aging by small chaperones: A trade-off with cancer?

Aging is a complex process accompanied by a decreased capacity of cells to cope with random molecular damages. Damaged proteins can form aggregates and have cytotoxic properties, a feature of many age-associated diseases. Small Hsps are chaperones involved in the refolding and/or disposal of protein aggregates. In Drosophila melanogaster, the mitochondrial DmHsp22 is preferentially upregulated during aging. Its over-expression results in an extension of lifespan (>30%) and an increased resistance to stress. Although DmHsp22 has a chaperone-like activity in vitro, additional mechanisms by which it may extend lifespan in vivo are unknown. Genome-wide transcriptional analysis and comparative mitochondrial proteomic analysis by MALDI-TOF were performed to unveil differences in long-lived DmHsp22 over-expressing flies. Flies over-expressing DmHsp22 display an upregulation of genes normally downregulated with age and involved in energy production and protein biosynthesis. Interestingly, DmHsp22 over-expression extended lifespan of normal fibroblasts by slowing the aging process. However, its expression also increased the malignant properties of human transformed cells. The delicate balance between beneficial and noxious effects of this small chaperone are discussed.

Proproliferative functions of Drosophila small mitochondrial heat shock protein 22 in human cells.

Aging is a complex process accompanied by a decreased capacity of cells to cope with random damages induced by reactive oxygen species, the natural by-products of energy metabolism, leading to protein aggregation in various components of the cell. Chaperones are important players in the aging process as they prevent protein misfolding and aggregation. Small chaperones, such as small heat shock proteins, are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, mitochondrial Hsp22 (DmHsp22), is localized in the mitochondrial matrix and is preferentially up-regulated during aging. Its overexpression results in an extension of life span (>30%) (Morrow, G., Samson, M., Michaud, S., and Tanguay, R. M. (2004) FASEB J. 18, 598-599 and Morrow, G., Battistini, S., Zhang, P., and Tanguay, R. M. (2004) J. Biol. Chem. 279, 43382-43385). Long lived flies expressing Hsp22 also have an increased resistance to oxidative stress and maintain locomotor activity longer. In the present study, the cross-species effects of Hsp22 expression were tested. DmHsp22 was found to be functionally active in human cells. It extended the life span of normal fibroblasts, slowing the aging process as evidenced by a lower level of the senescence associated beta-galactosidase. DmHsp22 expression in human cancer cells increased their malignant properties including anchorage-independent growth, tumor formation in nude mice, and resistance to a variety of anticancer drugs. We report that the DmHsp22 interacts and inactivates wild type tumor suppressor protein p53, which may be one possible way of its functioning in human cells.

The level of Hsp27 in lymphocytes is negatively associated with a higher risk of lung cancer.

Heat shock proteins (Hsps) can protect cells, organs, and whole organisms against damage caused by abnormal environmental hazards. Some studies have reported that lymphocyte Hsps may serve as biomarkers for evaluating disease status and exposure to environmental stresses; however, few epidemiologic studies have examined the associations between lymphocyte Hsps levels and lung cancer risk. We examined lymphocyte levels of Hsp27 and Hsp70 in 263 lung cancer cases and age- and gender-matched cancer-free controls by flow cytometry. Multivariate logistic regression models were used to estimate the association between lymphocyte Hsps levels and lung cancer risk. Our results showed that Hsp27 levels were significantly lower in lung cancer cases than in controls (16.5 vs 17.8 mean fluorescence intensity, P < 0.001). This was not observed for Hsp70 levels. Further stratification analysis revealed that lymphocyte Hsp27 levels were negatively associated with lung cancer risk especially in males and heavy smokers. There was a statistical trend of low odd ratios (95% confidence intervals) and upper tertile levels of Hsp27 [1.000, 0.904 (0.566-1.444) and 0.382 (0.221-0.658, P (trend) = 0.001) in males and 1.000, 0.9207 (0.465-1.822) and 0.419 (0.195-0.897, P (trend) = 0.036) in heavy smokers] after adjustment for confounding factors. These results suggest that lower lymphocyte Hsp27 levels might be associated with an increased risk of lung cancer. Our findings need to be validated in a large prospective study.

Proteomic characterization of mouse cytosolic and membrane prostate fractions: high levels of free SUMO peptides are androgen-regulated.

The prostate is a relatively homogeneous tissue that is highly specialized in synthetic and secretory functions. The frequency of malignant growth explains its great clinical significance. We used here a combination of subcellular fractionation, 1-DE (one-dimensional gel electrophoresis) protein separation and mass spectrometry, to establish a prostate protein expression profile in mice. Analysis of proteins present in cytosolic (C) and membrane (P) prostate fractions led to the identification of 619 distinct proteins. A majority of abundant proteins were found to compose the metabolism and protein synthesis machinery. Those identified also correspond to known endoplasmic reticulum and Golgi residents, chaperones and anterograde cargos. They included a series of proteins involved in exocytic/endocytic trafficking. Among the signaling proteins, we identified the ubiquitin-like peptides smt3. We showed that both free small ubiquitin-related modifier SUMO-2/3 and SUMO-1 levels are subject to tight control by the androgen 5alpha-dihydrotestosterone (DHT). By contrast with SUMO-2/3, free SUMO-1 peptides are particularly abundant in the prostate when compared with other tissues. Therefore, we report prostate protein expression profiles of cytosolic and membrane fractions in mice. Our data suggest that the identified free SUMO peptides play an important role in this secretory tissue.

Hepatic stress in hereditary tyrosinemia type 1 (HT1) activates the AKT survival pathway in the fah-/- knockout mice model.

BACKGROUND/AIMS: The AKT survival pathway is involved in a wide variety of human cancers. We investigated the implication of this pathway in hereditary tyrosinemia type 1 (HT1), a metabolic disease exhibiting hepatocellular carcinoma (HCC), despite treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexadione (NTBC) which prevents liver damage. HT1 is an autosomal recessive disorder caused by accumulation of toxic metabolites due to a deficiency in fumarylacetoacetate hydrolase (FAH), the last enzyme in the catabolism of tyrosine. METHODS: NTBC withdrawal in the murine fah(-/-) knockout model was used to analyze in vivo the correlation between pathophysiological, biochemical and histological features consistent with hepatocarcinogenesis and activation of the AKT survival pathway. RESULTS: The HT1 stress initiated by NTBC discontinuation causes a progressive increase of liver and kidney pathophysiology. A stable activation of the AKT survival pathway is observed in the liver but not in kidneys of fah(-/-) mice. Hepatic survival is reinforced by inhibition of mitochondrial-mediated apoptosis through inactivation of Bad and induction of BCl-X(L) and BCl-2. CONCLUSIONS: The chronic stress induced by liver disease in HT1 activates the AKT survival signal and inhibits intrinsic apoptosis to confer cell death resistance in vivo and favor hepatocarcinogenesis.

Rescue from neonatal death in the murine model of hereditary tyrosinemia by glutathione monoethylester and vitamin C treatment.

Hereditary tyrosinemia type 1 (HT1) is a recessive disease caused by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH) that catalyzes the conversion of fumarylacetoacetate (FAA) into fumarate and acetoacetate. In mice models of HT1, FAH deficiency causes death within the first 24h after birth. Administration of 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC) prevents neonatal death in HT1 mice, ameliorates the HT1 phenotype but does not prevent development of hepatocellular carcinoma later on. FAA has been shown to deplete cells of glutathione by forming adducts. We tested whether a combination of a cell membrane permeable derivative of glutathione, glutathione monoethylester (GSH-MEE) and vitamin C could provide an alternative effective treatment for HT1. GSH-MEE (10 mmol/kg/j)/vitamin C (0.5 mmol/kg/j) treatment was given orally to pregnant/nursing female mice. While FAH-/- pups died in absence of treatment, all FAH-/- pups survived the critical first 24h of life when the mothers were on the GSH-MEE/vitamin C treatment and showed normal growth until postnatal day 10 (P10). However, after P10, pups showed failure to thrive, lethargy and died around P17. Thus, GSH-MEE/vitamin C supplementation could rescue the mice model of HT1 from neonatal death but it did not prevent the appearance of a HT1 phenotype in the second week after birth.

Using lymphocyte and plasma Hsp70 as biomarkers for assessing coke oven exposure among steel workers.

BACKGROUND: Hsp70, an early-response protein induced when organisms are confronted with simple or complicated environmental stresses, can act as either a cellular protector or a danger signal. OBJECTIVES: The goal of this study was to evaluate levels of lymphocyte and/or plasma Hsp70 as biomarkers for assessing exposure response to complex coke oven emissions (COEs). METHODS: We recruited 101 coke oven workers and determined levels of polycyclic aromatic hydrocarbon (PAH) exposure, urinary 1-hydroxypyrene (1-OHP), genotoxic damage by comet assay and micronuclei test, and other markers of damage, including plasma malondialdehyde (MDA) and lactate dehydrogenase (LDH). These were compared to levels of lymphocyte (intra-cellular) and plasma (extracellular) Hsp70 using Western blots and enzyme-linked immunosorbent assays (ELISA), respectively. RESULTS: We observed a COEs-related dose-dependent increase in levels of DNA damage, micronuclei rate, MDA concentration, and LDH activity. Lymphocyte Hsp70 levels increased in the intermediate-exposure group (1.39 +/- 0.88) but decreased in the high-exposure group (1.10 +/- 0.55), compared with the low-exposure group. In contrast, plasma Hsp70 levels progressively increased as the dose of exposure increased. Negative correlations were seen between lymphocyte Hsp70 levels and olive tail moment and LDH activity in the intermediate- and high-exposure groups. However, we observed positive correlations between plasma Hsp70 levels and LDH activity in the low and intermediate groups. CONCLUSIONS: In workers exposed to COEs, high lymphocyte Hsp70 levels may provide protection and high plasma Hsp70 levels may serve as a danger marker. Larger validation studies are needed to establish the utility of Hsp70 as a response marker.

Association of hsp70 polymorphisms with risk of noise-induced hearing loss in Chinese automobile workers.

Severe noise exposure can induce heat shock proteins (Hsps), and exposure to moderate noise has been reported to confer protection against noise-induced damage to hearing. Whether there is any association of genetic variation in both constitutive and inducible hsp70 genes with noise-induced hearing loss (NIHL) is presently unknown. Using polymerase chain reaction-restriction fragment length polymorphism, we genotyped 3 polymorphisms (+190A/ B, +1267A/B, and +2437A/B) in the hsp70-1 (rs1043618), hsp70-2 (rs1061581), and hsp70-hom (rs2227956) genes, respectively, and investigated the associations of these polymorphisms with risk of developing NIHL in 194 automobile workers working in a similar noise environment as evaluated by audiological assessment. Multivariate logistic regression models were used to assess the associations with the risk genotypes, and Whap software was used to analyze their haplotypes. Our results showed that there was no statistically significant difference in the genotype and allele distributions of hsp70-1, hsp70-2, and hsp70-hom between the NIHL group and the normal group (P > 0.05) with and without adjustment for age, sex, smoking, history of explosive noise exposure, and cumulative noise exposure. However, haplotype analysis revealed that the Hap5 (ie, haplotype +190A/+1267B/+2437A) and Hap6 (ie, haplotype +190A/+1267B/+2437B) were significantly more frequent in the NIHL group than in the normal group (20/9, P = 0.022, and 7/0, P = 0.005, respectively). Compared with Hap1 (ie, +190A/+1267A/+2437A), Hap5 was associated with a nearly 3-fold increased risk of NIHL (adjusted odds ratio, 2.67; 95% confidence interval, 1.13-6.27). Seven of the NIHL patients had Hap6, but none of the controls had this haplotype. Our results suggest that some haplotypes of the hsp70 genes may be associated with a higher susceptibility to NIHL.