INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Atopy, a predisposition to produce specific IgE antibody in response to common inhaled allergens such as pollens or mites, is commonly associated with asthma, especially in childhood. The development of atopy and asthma are determined by multiple and interacting genetic and environmental influences. The former include genes that influence both the development and specificity of IgE responses and the presence of bronchial hyperresponsiveness; these are usefully reviewed elsewhere (1). Marked geographical differences and temporal changes have been observed in the prevalence of atopy and asthma, suggesting important environmental influences which may include intensity of exposure to relevant aeroallergens in early life (2). Opportunities to study interactions between these genetic and environmental effects are limited by the paucity of informative polymorphisms and by difficulties both in phenotype definition and quantification of relevant allergen exposure.

Occupational sensitization and asthma initiated by low- molecular-weight chemicals, in which a specific immunological response to a single chemical hapten can be studied within a defined population with well-characterised exposure, overcomes several of these difficulties. We have previously reported the risk of developing specific IgE antibody to a human serum albumin conjugate of the acid anhydride, trimellitic anhydride (TMA), to be increased in human leukocyte-associated antigen-DR3 (HLA-DR3)-positive individuals (3). Subsequent, unpublished analysis of this population suggested that the relative risk was greater among those who had experienced lower intensities of exposure to TMA.

We have now investigated a workforce exposed to another well-recognized chemical cause of sensitization and asthma, ammonium hexachloroplatinate (ACP). Occupational exposure to ACP dust may be followed by the development of specific IgE-associated sensitization, manifest as a positive skin prick test to a solution of ACP (4). In a high proportion of cases (5) this is accompanied by symptoms consistent with occupational asthma which, as demonstrated by inhalation testing, can be provoked by ACP. Previous studies of platinum refinery workers have shown an increased risk in those working in more heavily exposed jobs (6) and in cigarette smokers (5) with a possible interaction between these (6). Following our findings in acid anhydride workers (3) we hypothesized that the risk of occupational sensitization to ACP would be increased among HLA-DR3-positive individuals and the relative risk would be greater among those in jobs with lower intensities of exposure.

	METHODS

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We studied the current workforce (700 employees) of the largest platinum refinery in the world; in addition to process workers these included engineers and other technical staff. All workers are screened at first employment, and every 3 to 6 mo thereafter, by skin prick tests to identify the development of specific IgE to ACP; and at other times if they present with symptoms suggestive of allergy to ACP. We defined cases as all those who developed, during their employment at the refinery, an immediate skin prick test wheal with a diameter at least 3 mm greater than the negative control (saline) to ACP in a concentration of less than 10³ g/L. For each case one or more referents, matched on age, race (black/white), employment duration, and job title (as an index of intensity of exposure to ACP), was selected at random from those who had not developed a skin test response to ACP at a maximal concentration of 10³ g/L. If eligible, each referent could be used more than once. The distribution of HLA phenotypes in cases and referents was compared.

Prior to analysis, workers were allocated by their occupational physician (R.D.) to one of two categories of ACP exposure (high versus low) using a matrix of work area and job title. Employees working in process areas where the concentration of complex platinum salts was greater than 10 g/L were allocated to the high-exposure group. Those who regularly worked in all areas of the refinery with high potential for exposure to process liquors and/or residues (determined by job title) were also allocated to this group. The remaining employees were categorized as experiencing low exposures. Routine exposure measurements, using personal gravimetric sampling, indicated that employees were rarely exposed to time-weighted average concentrations of airborne ACP greater than the current recommended exposure limit of 2 µg/m³.

Genomic DNA Preparation

Genomic DNA was extracted using a high-salt technique with minor modifications as previously described (7, 8).

Polymerase Chain Reaction-Sequence-specific Oligonucleotide (PCR-SSO) Analysis

HLA-typing was performed without knowledge of the subject's case status. The second exons of the HLA-DRB1, -DQA1, -DQB1, and -DPB1 chains were amplified by PCR using the specific generic HLA primer pairs supplied by the British Society for Histocompatibility and Immunogenetics (BSHI) (Dr. S. Martin, St. Mary's Hospital, Manchester, UK).

Genomic DNA (1 mg) was mixed with 1× PCR buffer containing 1.5 mM MgCl₂ (Boehringer Mannheim, Lewes, E. Sussex, UK), 0.2 mM each deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine triphosphates (Pharmacia, St. Albans, Herts, UK), 1 mg of each primer, and 0.3 U Taq DNA polymerase (Boehringer Mannheim) in a final volume of 50 ml. PCR amplification was automated with a thermal cycler (Techne, Cambridge, UK). The initial denaturation was for 3 min at 95° C, followed by 30 cycles of 95° C for 1.3 min, 55° C for 2 min, and 72° C for 2 min. The final extension was maintained for 10 min. Ten percent of the reaction volume was then electrophoresed on a 1.2% agarose gel containing 0.1% wt/vol ethidium bromide (Sigma Ltd., Poole, Dorset, UK). The amplified DNA was denatured and transferred to Hybond-N+ nylon membranes (Amersham plc, Amersham, Bucks, UK) by the Southern blotting procedure (9). The membranes were washed in 1× saline sodium citrate (SSC) solution and cross-linked by ultraviolet radiation, using an automatic cross linker (Hybaid, Teddington Middlesex, UK).

The 5' biotinylated HLA-DRB1, -DQA1, -DQB1, and -DPB1 sequence-specific oligonucleotide (SSO) probes (BSHI) were each hybridized with the cross-linked membranes. The membranes were prehybridized as described for enhanced chemiluminescent (ECL) 3' oligolabeling and detection (Amersham) at 48° C or 52° C (17 mers and 21 mers, respectively). The oligonucleotide probe was then added and hybridized with the membrane for 2 h. The membranes were washed and blocked as described prior to incubation with streptavidin-horseradish peroxidase conjugate (Amersham) for 30 min at room temperature. Signal generation and detection were carried out as described by the manufacturer and autoradiography was performed by exposure to Kodak XAR-5 film (Sigma Ltd).

Polymerase Chain Reaction-Sequence-specific Primer (PCR-SSP) Analysis

To confirm and specify the identification of HLA-DRB3, -DRB4, and -DRB5, the samples were reanalyzed using the technique of PCR-SSP using the appropriate sequence-specific primers as previously described (10). All PCR reactions were performed in a final reaction volume of 13 µl excluding 10 µl of light mineral oil overlay as previously described (4). The PCR reaction mixtures contained 67 mM Tris base pH 8.8, 16.6 mM NH₄SO₄, 2 mM MgCl₂, 0.01% (vol/vol) Tween 20, 200 µM each deoxyribonucleoside triphosphate (dNTP) (deoxyadenosine, deoxyguanosine, deoxyadenosine, and deoxythymidine triphosphate), 1 to 4 µM each allele-specific primer, 0.1 µM each control primer (63, 64; Reference 10), between 0.1 and 0.01 µg DNA, and 0.1875 U Taq DNA polymerase (Boehringer Mannheim). The sequence of addition of buffer mixture to specific primers has previously been described (10). PCR amplifications were carried out in an MJ Research PTC-192V machine (Genetic Research Instrumentation Ltd, Dunmow, Essex, UK).

The cycling parameters were as follows: 1 min at 96° C; 5 cycles of 25 s at 96° C, 45 s at 70° C, 45 s at 72° C; 21 cycles of 25 s at 96° C, 50 s at 65° C, 45 s at 72° C; 4 cycles of 25 s at 96° C, 60 s at 55° C, 120 s at 72° C. The plates were sealed and the lid of the thermocycler was adjusted to ensure firm and even pressure on the plate within the machine.

The PCR products were electrophoresed (200 V, 15 min) in 1% agarose gels containing 0.5 µg/ml ethidium bromide after the addition of 5 µl of Orange G loading buffer (4) to each well of the PCR plate and the results photographed.

Subjects

All employees were male. Forty-five cases were identified and matched with 66 referents. Sixteen cases were matched with one referent, 24 with two, and five with three. Eighty-two employees (74%) were in high-exposure jobs. There was a higher proportion of white employees among those in low-exposure jobs (62% versus 44%), although the difference was not statistically significant (p = 0.13). There was no difference in smoking within the exposure categories (52% low, 48% high, p = 0.83). Blood was unavailable for two referents and it was not possible to extract DNA for a further seven referents and one case, leaving a total of 101 employees for typing. Details of the subjects used in the analysis are shown in Table 1. Cases and their referents were closely matched on age, race, duration, and intensity of ACP exposure.

Statistical Analysis

Ten HLA-DR phenotypes were examined. In addition we explored DQA, DQB, and DPB phenotypes. Differences between the cases and their matched referents were analyzed using conditional logistic regression, with results expressed as odds ratios (OR) and 95% confidence intervals (95% CI). Interactions between phenotype and exposure intensity were analyzed by including an interaction term in each model. All analysis was done using SAS (SAS Institute Inc., Cary, NC) and Egret (SERC, Seattle, WA) statistical software.

	RESULTS

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The distribution of all tested phenotypes by case status is summarized in Table 2. There were no significant differences (p > 0.05) between cases and referents in HLA-DR1, -DR2, -DR4, -DR5, -DR7, -DR8, -DR9, or -DR10 phenotypes, or in HLA-DQA, -DQB, and -DPB phenotype frequencies. HLA-DR3 was more common among cases (OR 2.3) (Table 3); the odds ratio associated with this phenotype was higher in those with low exposure (OR infinite) than with high (OR 1.6). It was not possible to estimate the strength of interaction between HLA-DR3 and exposure as there were no HLA-DR3-negative cases with a matched HLA-DR3-positive control in the low-exposure group. HLA-DR6 was less common among the cases (OR 0.4), an association which was also stronger in the low-exposure group (OR 0.1 versus 0.5; p = 0.29). The rate of smoking was significantly higher among cases (OR 3.9); especially those with low exposure (OR 9.2 versus 3.1; p = 0.35).

The association between HLA-DR6 phenotype and ACP sensitivity was stronger among white workers, OR 0.2 (0.03, 0.7) versus OR 0.6 (0.2, 1.9); p = 0.14. Within each race, this relationship was again stronger in the low-exposure group. There was no difference in the association between HLA-DR3 and ACP sensitivity by race.

We identified no common amino acid motifs in HLA-DR molecules among the cases. Fifteen subjects were both HLA-DR2- and -DR6-positive, all of whom were referents. All 44 cases who were typed were either HLA-DR3-positive, -DR2-negative or -DR6-negative. Conversely, 48 (84%) of the referents were either HLA-DR3-negative, -DR2-positive, or -DR6-positive.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our findings indicate that HLA phenotype is a significant determinant of sensitization to the inhaled low-molecular-weight chemical ACP, a well-described cause of asthma. We have also for the first time shown that the strength of this association varies with intensity of exposure to the sensitizing agent, being greater in those exposed to lower concentrations of ACP. Interestingly we also found that the relative risk in cigarette smokers was greater in those in the low-exposure group. These observations seem unlikely to be the result of bias; although there was some confounding of estimated exposure by race (the proportion of white employees was greater in low-exposure jobs) this did not explain the observed interaction between HLA phenotype and exposure with the risk of sensitization to ACP.

We also observed in this workforce that there was a deficit of HLA-DR6 individuals in those sensitized to ACP and that the relative risk was also stronger among those with low-exposure jobs. All cases were either HLA-DR3-positive, or DR6- or DR2-negative; 84% of their referents were HLA-DR3 negative or DR6- or DR2-positive. The relationships with DR6 and DR2 were not, however, part of our prior hypothesis and although interesting require confirmation in a separate population with ACP exposure.

Our estimate of the risks associated with particular phenotypes may be compared with other known risk factors, including the intensity of exposure to platinum salts and cigarette smoking. An earlier study from a different refinery calculated a hazard ratio of 6.2 for sensitization among those in high- exposure jobs, defined as those that included some exposure to airborne platinum salts at above the threshold limit value of 2 µg/m (6). This figure is considerably higher than our risk estimates for HLA-DR3, even among employees with low exposure. Similarly our results suggest that the proportions of sensitization that are attributable to HLA-DR3 phenotype and to cigarette smoking are, respectively, approximately 25% and 51%. Together, these confirm the primary importance of environmental determinants of occupational sensitization.

There is now consistent evidence that asthma and sensitization to low-molecular-weight chemicals encountered at work is influenced by HLA polymorphisms. In addition to our observations in TMA workers, Bignon and colleagues (11) have reported that asthma induced by toluene diisocyanate occurs less frequently in workers with HLA-DQB1*0501 and more frequently in those with HLA-DQB1*0503 alleles. In a subsequent study of a different population Balboni and coworkers (12) confirmed these relationships and suggested that aspartic acid in position 57 in the hypervariable region of the  chain of DQB1*0503 as opposed to valine in position 57 of DQB1*0501 might explain the susceptibility to the development of disease. We observed no distinguishing amino acid motif to identify susceptibility to the development of specific IgE to ACP in our study population.

Our findings, if confirmed, have important implications. They indicate that, in the case of IgE-sensitization to a low-molecular-weight chemical, the influence of genetic susceptibility seems greatest among those with least exposure to the causative allergen. An interaction between competing environmental and genetic causes of disease has been reported in carcinogenesis where the risk of lung cancer associated with the Dra I polymorphism of the C7P2E1 gene was greater among those smoking few cigarettes (13). Similar modification of the response to environmental allergens has also been suggested in studies of childhood sensitization to ubiquitous aeroallergens; in a cohort of German children, for example, sensitization to Dermataphagoides and cat allergens among those with a family history of atopy was achieved at a lower level of infant exposure to the relevant allergen (14). Our results, analyzing a specific phenotype, imply that as interventions to control exposure in the workplace are introduced, genetic polymorphisms will increasingly determine the risk of disease. More broadly, if sensitization and asthma caused by agents inhaled at work is a relevant model for atopy and asthma outside the workplace (15), then genetic susceptibility will increasingly determine their development in environments where allergen exposure (for example to Dermataphagoides species) is low, or where preventive measures to reduce it have been undertaken.