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Keywords:

  • allergic bronchopulmonary aspergillosis;
  • asthma;
  • cystic fibrosis;
  • HLA

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease that affects both patients with cystic fibrosis (CF) and those with asthma. HLA-DRB1 alleles have previously been associated with ABPA–CF susceptibility; however, HLA-DQB1 allele associations have not been clearly established. The aim of the present study was to investigate HLA class II associations in patients with ABPA–CF and determine their roles in susceptibility or protection. Patients with ABPA–CF, patients with CF without ABPA, patients with asthma without ABPA (AST), and healthy controls were included in this study. DNA was extracted by automatic extractor. HLA-DRB1 and -DQB1 genotyping was performed by the Luminex PCR-SSOP method (One Lambda, Canoga Park, CA, USA). Allele specific PCR-SSP was also performed by high-resolution analysis (One Lambda). Statistical analysis was performed with SSPS and Arlequin software. Both HLA-DRB1*5:01 and -DRB1*11:04 alleles occurred with greater frequency in patients with ABPA–CF than in those with AST and CF and control subjects, corroborating previously published data. On the other hand, analysis of haplotypes revealed that almost all patients with ABPA–CF lacking DRB1*15:01 or DRB1*11:04 carry either DRB1*04, DRB1*11:01, or DRB1*07:01 alleles. In the HLA-DQB1 region, the HLA-DQB1*06:02 allele occurred more frequently in patients with ABPA–CF than in those with AST and CF and healthy controls, whereas HLA-DQB1*02:01 occurred less frequently in patients with ABPA–CF. These data confirm that there is a correlation between HLA-DRB1*15:01, –DRB1*11:04, DRB1*11:01, –DRB1*04 and –DRB1*07:01 alleles and ABPA–CF susceptibility and suggest that HLA-DQB1*02:01 is an ABPA–CF resistance allele.

Abbreviations
ABPA

allergic broncho-pulmonary aspergillosis

A. fumigates

Aspergillus fumigates

AST

patients with asthma without ABPA

CF

cystic fibrosis

CFTR

CF transmembrane conductance regulator

CS

unrelated healthy Caucasoid blood donors (controls)

HLA

human leukocyte antigen

IL

interleukin

SSOP

sequence-specific oligonucleotide primers

SSP

sequence-specific primers

Cystic fibrosis (MIM 219700) is the most common autosomal recessive disease in Caucasians [1]. Chronic lung disease, pancreatic insufficiency and male infertility are the most characteristic clinical features. All of these phenotypic abnormalities are caused by mutations in the CFTR gene (MIM 602421). A spectrum of CFTR mutations in patients with CF from the region of Murcia (southeast of Spain) has previously been reported [2, 3].

On the other hand ABPA, a hypersensitivity lung disease that affects both patients with CF and those with asthma, is caused by colonization of the airways with the fungus Aspergillus fumigatus [4, 5]. ABPA affects approximately 1–2% of patients with AST and 7–9% of those with CF [6]. The clinical features of ABPA include asthma, pulmonary infiltrates, bronchiectasis and pulmonary fibrosis. The immune and inflammatory responses against A. fumigatus antigens are characterized by increases in total serum IgE, specific IgE and IgG antibodies and precipitating antibodies and eosinophilia [7]. T cell reactivity in ABPA is characterized by the presence of CD4+ T cells producing IL-4 and IL-5 cytokines [8-10].

Associations between HLA class II antigen purified allergens and IgE responses have previously been reported [11-16]. Indeed, HLA-DRB1 alleles have previously been associated with ABPA susceptibility, although HLA-DQB1 allele associations have not been clearly established [17, 18].

Our aim was to study HLA class II allele frequencies in our patients with ABPA–CF and compare their allele frequencies with those of patients with CF without ABPA, those with AST and healthy subjects to determine the role of various alleles in susceptibility or protection.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Patients and controls

Patients with ABPA–CF (n = 38), CF without ABPA (n = 46) and AST (n = 306) included in this study were recruited at the University Hospital Virgen de la Arrixaca from the Murcia region, in the southeast of Spain. CF mutational analysis was performed by the genetic service of our hospital, as previously reported [2, 3]. Patients with AST were diagnosed as previously reported [15, 16]. The control group comprised 176 unrelated healthy Caucasoid blood donors (CS) living in the same area. Patients with ABPA fulfilled the criteria for this diagnosis, as outlined by Patterson et al. [17]. ABPA was diagnosed by the presence of recurrent wheezing, chest radiographic infiltrates, peripheral blood eosinophilia, immediate A. fumigatus skin reactivity, positive precipitating antibodies against A. fumigatus antigens, increased serum total IgE concentrations of greater than 1000 IU/mL and IgE and IgG anti-A. fumigatus antibodies. Prior to their inclusion in this study, all subjects gave their informed consent to the collection and storage of blood, isolation of DNA and determination of gene polymorphisms. The study was approved by the Local Medical Ethics Committee.

DRB1 and DQB1 genotyping

DNA was extracted by a Maxwell16 extractor (Promega Madison, WI, USA) by a previously published method [18]. HLA-DRB1 and –DQB1 genotyping was performed by Luminex PCR-SSOP methodology (One Lambda), according to the manufacturer's recommended procedure, as previously published [19]. In addition, allele specific PCR-SSP (One Lambda) was performed by high-resolution analysis, by a previously published method [20].

Statistical analysis

Statistical analysis of distribution of allele frequencies between groups was performed by SSPS v15.0 and Arlequin V2.0 (University of Geneva) software, as previously described [18, 20]. Categorical data were analyzed using Fisher's exact test and the likelihood ratio χ2 test. P-value < 0.05 was considered as significant. P values were corrected by Bonferroni correction (Pc), as previously described [18].

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Allele frequencies in AST, CF and healthy control groups were very similar, no significant differences being found between these groups.

However, both HLA-DRB1*15:01 (Pc = 0.03) and –DRB1*11:04 (borderline, Pc = 0.07) alleles occurred with greater frequency in patients with ABPA–CF than in controls, patients with CF and patients with AST, corroborating the data previously published by Chauhan et al. [12] (Table 1).

Table 1. HLA-DRB1 and –DQB1 alleles in patients with ABPA–CF, AST and CS
AlleleCS frequency (2n = 352)ABPA–CF frequency (2n = 76)AST frequency (2n = 612)CF frequency (2n = 92)Pc
  1. a

    Patients with ABPA–CF versus AST, patients with CF and controls-CS.

  2. b

    Patients with ABPA–CF versus AST, patients with CF and CS.

  3. c

    Patients with ABPA–CF versus AST, patients with CF and CS.

  4. d

    Patients with ABPA–CF versus AST, patients with CF and CS. NS, not significant; Pc, corrected P.

DRB1*01:010.075400.076210.056890.06712NS
DRB1*01:020.059520.076210.048740.06712NS
DRB1*01:030.015870.000000.007860.00765NS
DRB1*03:010.123020.115350.151920.13195NS
DRB1*03:020.003970.000000.007860.00000NS
DRB1*04:010.035710.000000.056890.03426NS
DRB1*04:020.027780.038790.033830.03426NS
DRB1*04:030.015870.000000.033830.02112NS
DRB1*04:040.019840.076210.029100.02112NS
DRB1*04:050.011900.000000.018630.00000NS
DRB1*04:070.007940.000000.007860.00000NS
DRB1*07:010.178570.230730.163730.176750.04a
DRB1*08:010.011900.000000.029100.02112NS
DRB1*08:030.003970.000000.018630.00765NS
DRB1*08:040.003970.000000.018630.00765NS
DRB1*09:010.007940.000000.007860.00765NS
DRB1*10:010.007940.000000.018630.00765NS
DRB1*11:010.031750.038750.029100.03426NS
DRB1*11:020.031750.000000.029100.03426NS
DRB1*11:030.007940.000000.007860.00765NS
DRB1*11:040.031750.059520.033830.02112NS
DRB1*12:010.011900.000000.018630.00765NS
DRB1*13:010.083330.115950.091740.08635NS
DRB1*13:020.051590.038790.056890.05547NS
DRB1*13:030.015870.000000.018630.00765NS
DRB1*14:010.003970.000000.007860.00765NS
DRB1*15:010.051590.153800.061620.055470.03b
DRB1*15:020.019840.000000.018630.00765NS
DRB1*16:010.019840.000000.018630.00765NS
DQB1*02:010.123020.083330.141860.131950.04c
DQB1*02:020.174600.230730.160180.17675NS
DQB1*03:010.166670.125610.160180.17675NS
DQB1*03:020.111110.115350.124360.09762NS
DQB1*03:030.011900.038790.007860.00765NS
DQB1*04:010.003970.000000.000000.00000NS
DQB1*04:020.019840.000000.018630.02112NS
DQB1*05:010.158730.153810.151920.15422NS
DQB1*05:020.023810.000000.018630.00765NS
DQB1*05:030.003970.000000.007860.00765NS
DQB1*06:010.019840.000000.018630.02112NS
DQB1*06:020.047620.153800.061620.055470.03d
DQB1*06:030.087300.115950.091740.08635NS
DQB1*06:040.039680.038790.056890.05547NS
DQB1*06:090.007940.000000.000000.00000NS

On the other hand, analysis of haplotypes revealed that almost all patients with ABPA–CF lacking DRB1*15:01 or DRB1*11:04 carried either DRB1*04, DRB1*11:01 or DRB1*07:01 alleles (Pc = 0.04, ABPA–CF vs AST).

Thus, 84% of patients with ABPA–CF carried either DRB1*15:01, DRB1*11:04, DRB1*11:01, DRB1*07:01, and/or DRB1*04 alleles at a significantly higher frequency than was found in controls, patients with CF and patients with AST (Table 1). The DRB1*03:01 allele frequency was less in patients with ABPA–CF than in controls, patients with CF and patients with AST, although this difference was not significant.

There were no significant differences between the compared groups in the remaining HLA-DRB1 alleles. The DRB1*15:03 allele reported by Chauhan et al. [12] was not found in any of our controls or patients.

The HLA-DQB1*06:02 allele occurred with greater frequency in patients with ABPA–CF than in patients with AST, patients with CF and healthy controls; this allele was the most frequently occurring in patients with ABPA–CF in contrast to controls, patients with CF and AST (Pc = 0.03 ABPA–CF vs AST, CS). However, the HLA-DQB1*02:01 allele occurred less frequently in patients with ABPA–CF than in the other groups (Pc = 0.04 ABPA–CF vs. AST, CF, CS; Table 1).

HLA-DRB1*15:01 has strong linkage with HLA-DQB1*06:02. Therefore, the observed high frequency of this HLA-DQB1 allele may simply reflect the high frequency of the DRB1*15:01 allele in patients with ABPA–CF.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This study provides new information about the HLA-DQ2 serological subtypes reported by Chauhan et al. [12], namely the HLA-DQB1*02:02 subtype, an eventual allele for ABPA–CF susceptibility and HLA-DQB1*02:01, a possible allele of ABPA–CF protection. The difference between DQB1*02:01 and DQB1*02:02 is in exon 3 (amino acid 135). The DQB1*02:01 allele is genetically linked to DQA1*05:01 and has classically been associated with celiac disease, Type 1 diabetes and other autoimmune diseases. However, DQB1*02:02 is linked to several DQA1 alleles, namely DQA1*02:01 and DQA1*03:03. Thus, in future studies we will investigate other HLA genes to clarify other possible associations. In addition, because ABPA is an uncommon complication of CF, it will also be important to further investigate and corroborate these interesting findings with a larger number of patients in the future.

We found no differences between the groups used as comparison controls, which consolidates our findings. Our findings allow us to both corroborate and rule out partnerships with primary genetic pathology in patients with CF. With regard to patients with asthma, they allow us to discard possible associations with other allergic pulmonary pathology and, by making comparisons with healthy subjects, to determine general population frequencies.

In this context, several reports have shown that a strong Th2 response to A. fumigatus antigens, as indicated by prominent eosinophil infiltration, could be responsible for development of ABPA [21, 22]. Thus, it is possible that particular HLA class II alleles play critical roles in the outcome of T-cell responses (Th1 vs Th2) to A. fumigatus antigens. Thus, patients with CF but without ABPA who lack permissive alleles possibly have Th1 type responses against the fungus A. fumigates, which would prevent colonization of the lung and development of ABPA. The opposite situation would occur in patients with ABPA–CF and susceptibility alleles; they mount a Th2 type response [11, 15]. In this context, other authors have also demonstrated that altered T cell receptor-mediated signals can lead to altered T lymphocyte phenotypes [23]. This does not mean that a susceptibility allele alone can cause ABPA; however, these alleles could influence the outcome of exposure to A. fumigatus.

In conclusion, these data corroborate previous studies showing correlations between HLA-DRB1*15:01, –DRB1*11:01, –DRB1*11:04, –DRB1*07:01, –DRB1*04 alleles, and ABPA–CF susceptibility. Indeed, our data show that HLA-DQB1*02:01 is a possible ABPA–CF resistance allele.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This work was possible in part thank to technical support from projects from Fondo de Investigación Sanitaria (FIS) (PI11/02686) (CIBERehd) funded by the Instituto de Salud Carlos III, Spain and Seneca Foundation No. 04487/GERM/O6 y CajaMurcia.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

None of the authors has a conflict of interest to disclose. We confirm that we have read the journal's position on issues involved in ethical publication and we affirm that this report is consistent with those guidelines.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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