A major genetic determinant of autoimmune diseases is associated with the presence of autoantibodies in hypersensitivity pneumonitis

Study population

A retrospective review of adult patients (age ≥18 years) referred to the ILD clinic at the National Institute of Respiratory Diseases (Mexico City, Mexico) was conducted, and samples with enough DNA quality from 170 patients with a confirmed diagnosis of hypersensitivity pneumonitis during the period 2003–2018 were included (figure 1). Since the year 2000, DNA samples for genetic studies are obtained from patients with ILD, with clear explanation, and signed informed consent is duly collected.

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FIGURE 1

Inclusion of hypersensitivity pneumonitis with and without autoantibodies (HPAbs⁺ and HPAbs⁻, respectively), showing exclusion rates for the different analyses. PFT: pulmonary function test.

All patients had at least three prior generations born in Mexico (parents and grandparents) and were considered as Mexican mestizo. We have previously demonstrated that this criterion is a good proxy of Mexican ancestry evaluated by ancestry-informative markers [12].

Hypersensitivity pneumonitis was diagnosed according to previously described criteria [13, 14], as follows. 1) Symptoms and pulmonary function tests alterations compatible with ILD; 2) high-resolution computed tomography (HRCT) showing poorly defined nodules, ground-glass attenuation and air trapping in expiration (80% of the HPAbs⁺ and 54% of the HPAbs⁻ also showed alterations compatible with fibrosis, such as irregular linear opacities and traction bronchiectasis and occasionally cystic lesions); 3) bronchoalveolar lavage lymphocytosis >30%; and 4) biopsy (performed in 30% of the HPAbs⁺ and 28% of the HPAbs⁻) compatible with hypersensitivity pneumonitis. A multidisciplinary team confirmed the diagnosis.

Clinical data were obtained from the medical records. The variables collected included demographical data, comorbidities, tobacco smoking history, environmental antigen exposure assessment, clinical laboratory studies, pulmonary function tests (including forced vital capacity, diffusing capacity of the lung for carbon monoxide (D_LCO) and 6-min walk test) and mortality from all causes at 1-year follow-up.

We considered hypersensitivity pneumonitis with positive serology (HPAbs⁺) as positivity of at least one of the following antibodies: antinuclear antibodies (ANAs) with a specific pattern of connective tissue disease of any kind (cytoplasmic, nucleolar, centromere); ANA with homogeneous pattern, fine or coarse mottle; rheumatoid factor three or more times the upper normal limit (20 IU·mL⁻¹); anti-cyclic citrullinated peptide ≥20 RU·mL⁻¹ and/or at least one antibody in the autoimmunity profiles for Sjögren's syndrome (anti-Ro (SSA), anti-La (SSB)). These autoantibodies were analysed in all the patients. Additionally, autoantibodies associated with systemic sclerosis (anti-Scl-70), vasculitis (anti-neutrophil cytoplasmic antibody (ANCA)) and anti-DNA were examined in 88% of the HPAbs⁺ and 55% of the HPAbs⁻. No patient met classification criteria for connective tissue diseases according to the American College of Rheumatology/European League Against Rheumatism [15–17].

The patients were classified according to the presence of autoantibodies present in serum (HPAbs⁺ versus HPAbs⁻) (table 1).

Ethics statement

This study was approved by the institutional committee for science and ethics of the Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER; Mexico City, Mexico) (approbation codes: B20-15 and C60-17).

Genomic DNA isolation

Peripheral blood (7 mL) was obtained by venipuncture and was collected in a tube with EDTA as an anticoagulant for subsequent DNA extraction. Samples were obtained at the time of diagnosis, and patients were not receiving corticosteroids or immunosuppressive drugs.

The blood samples were handled in the HLA laboratory of INER to obtain genomic DNA employing a commercial kit (BDtract Genomic DNA Isolation Kit; Maxim Biotech, San Francisco, CA, USA). The DNA was quantified by ultraviolet spectrophotometry using a NanoDrop 2000 device (Thermo Scientific, Waltham, MA, USA). Contamination with organic compounds and proteins was defined by measuring the ratio absorbance at 280 nm and 260 nm. Samples were considered of good quality when the ratio was ∼1.8.

HLA class II molecular typing

Molecular typing for DRB loci and DQB1 locus was done using a PCR sequence-specific primers (PCR-SSP) technique using Micro SSP™ HLA DNA Typing Trays (One Lambda, Canoga Park, CA, USA). Unlike other PCR-based methods, the SSP methodology employed discriminates between the different alleles during the PCR process. The amplified DNA fragments are separated by agarose gel electrophoresis and visualised by staining and exposure to ultraviolet light. The interpretation of PCR-SSP results is based on the presence or absence of a specific amplified DNA fragment. Since amplification during the PCR reaction may be adversely affected by various factors such as pipetting errors, poor DNA quality and presence of inhibitors, an internal control primer pair was included in every PCR reaction. The control primer pair amplifies a conserved region of the human β-globin gene, which is present in all human DNA samples and is used to verify the integrity of the PCR reaction.

Initially, HLA-DRB typing was performed by low-resolution modality, which includes DRB1, DRB3, DRB4 and DRB5 alleles in 24 independent reactions. The DRB loci allele group covers DRB1*01, *03, *04, *07, *08, *11, *12, *13, *14, *15 and *16 as well as DRB3*02, DRB3*03, DRB4*01 and DRB5*01 specificities (DR51, DR52 and DR53 serological equivalents). High resolution for HLA-DRB1*03 was performed with an additional panel of 16 primer pairs to establish allele discrimination. For HLA-DQB1 typing, eight primer pairs were used, while the HLA-DQB1 high-resolution kit used to achieve DQB1 amplification, which includes 55 alleles for DQ6, DQ3, DQ4, DQ5 and DQ2.

All amplifications were performed using the Kapa Taq HotStart PCR kit (Kapa Biosystems, Cape Town, South Africa). PCR-SSP products were electrophoresed in 3% agarose gels stained using MIDORI Green Advanced DNA Stain (Nippon Genetics Europe, Dueren, Germany). Interpretation of typing results was made using the assistance of the HLA Fusion Software versus 4.3 (One Lambda).

Statistical analysis

The statistics programme SPSS (version 21; SPSS, Chicago, IL, USA) was used to describe the study population and determine the median, minimum, and maximum values for each variable and compared using a Mann–Whitney U-test. Continuous variables were reported as mean±sd and analysed using a t-test. Categorical variables were reported as counts and percentages and contrasted using Fisher's exact test.

Allele and haplotype frequencies of HLA were determined by direct counting. The observed and expected HLA class II alleles (each locus) tested for Hardy–Weinberg equilibrium using a conventional Fisher's exact test. The associations were evaluated by Fisher's exact two-tailed test, with a statistical significance value of p<0.05. Bonferroni correction was performed considering the 20 alleles identified in both loci and the 22 haplotypes found in the whole population. Odds ratios and 95% confidence intervals were calculated using 2×2 contingency tables comparing both groups by alleles and haplotypes reported using the Epi Info v4.0.1 [18]. Finally, the haplotypes were constructed employing Arlequin v3.1 software [19] using the maximum likelihood method, with an iterative expectation maximisation algorithm.

To evaluate the strength of association between mortality and the risk allele, we first estimated a crude odds ratio (cOR) using univariate regression analysis; the p-value was estimated with the Wald test, and then we estimated an adjusted odds ratio (aOR) with a multivariate logistic regression analysis that includes only variables with p<0.05, due to the small sample size. No imputation procedures were performed to handle missing data.