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Arquivos de Gastroenterologia

Print version ISSN 0004-2803On-line version ISSN 1678-4219

Arq. Gastroenterol. vol.56 no.2 São Paulo Apr./June 2019  Epub Aug 26, 2019

https://doi.org/10.1590/s0004-2803.201900000-29 

ORIGINAL ARTICLE

HLA-DRB1 GENE POLYMORPHISMS IN PEDIATRIC PATIENTS WITH TYPE 1 AUTOIMMUNE HEPATITIS AND TYPE 1 AUTOIMMUNE HEPATITIS OVERLAP SYNDROME WITH AUTOIMMUNE CHOLANGITIS

Polimorfismos do gene HLA-DRB1 em pacientes pediátricos com hepatite autoimune tipo 1 e hepatite autoimune associada à colangite autoimune

Monique Ellen Gervásio NUNES1 
http://orcid.org/0000-0002-4291-9221

Daniela Valadão ROSA1 
http://orcid.org/0000-0001-8868-7895

Eleonora Druve Tavares FAGUNDES2 
http://orcid.org/0000-0002-5671-9570

Alexandre Rodrigues FERREIRA2 
http://orcid.org/0000-0001-6749-8980

Débora Marques de MIRANDA1 
http://orcid.org/0000-0002-7081-8401

Priscila Menezes FERRI LIU2 
http://orcid.org/0000-0002-8608-8503

1Universidade Federal de Minas Gerais, Faculdade de Medicina, Laboratório de Medicina Molecular, Belo Horizonte, MG, Brasil.

2Universidade Federal de Minas Gerais, Hospital das Clínicas, Serviço de Gastroenterologia Pediátrica, Belo Horizonte, MG, Brasil.


ABSTRACT

BACKGROUND:

Autoimmune hepatitis (AIH) is a rare chronic inflammatory liver disease associated with a loss of immunological tolerance to self-antigens. Susceptibility to AIH is partially determined by the presence of genes related to human leukocyte antigen (HLA), mainly allelic variants of DRB1.

OBJECTIVE:

The purpose of this study was to investigate the frequencies of the polymorphisms in HLA-DRB1 gene in children and adolescents with type 1 AIH and type 1 AIH overlap syndrome with autoimmune cholangitis (overlap syndrome, OS) in comparison to healthy sex and age-matched individuals (control group).

METHODS:

This is a cross-sectional study of 25 pediatric patients diagnosed with type 1 AIH and 18 with OS. Fifty-seven healthy individuals were included as controls. The polymorphisms of the HLA-DRB1 gene were evaluated by PCR and included HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, and HLA-DRB1*13.

RESULTS:

Our results showed that the presence of the allele HLA-DRB1*13 increased the chance of autoimmune cholangitis (OR=3.96, CI 1.07 to 14.61, P=0.04). The HLA-DRB1*04 and HLA- DRB1*07 have no association with the AIH and autoimmune cholangitis in a young sample.

CONCLUSION:

This work demonstrates an association of the main polymorphisms in the HLA-DRB1 gene to AIH with or without cholangitis in a Brazilian sample.

HEADINGS: Autoimmune hepatites; Sclerosing cholangitis; HLA-DRB1 chains; Genetic polimorphism

RESUMO

CONTEXTO:

Hepatite autoimune (HAI) é uma doença hepática inflamatória crônica, rara, associada à perda da tolerância imunológica aos auto-antígenos. A susceptibilidade à HAI é parcialmente determinada pela presença de genes relacionados ao antígeno leucocitário humano (HLA), principalmente variantes alélicas do DRB1.

OBJETIVO:

O objetivo deste estudo foi investigar a frequência de polimorfismos no gene HLA-DRB1 em crianças e adolescentes com HAI tipo 1 e HAI tipo 1 associada à colangite autoimune, em comparação com indivíduos saudáveis pareados por sexo e idade (grupo controle).

MÉTODOS:

Este é um estudo transversal de 25 pacientes pediátricos com diagnóstico de HAI tipo 1 e 18 com HAI associada à colangite autoimune. Cinquenta e sete indivíduos saudáveis foram incluídos como controles. Os polimorfismos do gene HLA-DRB1 foram avaliados por PCR e incluíram HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07 e HLA-DRB1*13.

RESULTADOS:

Nossos resultados mostraram que a presença do alelo HLA-DRB1*13 aumentou a chance de colangite autoimune (OR=3,96; IC 1,07 a 14,61; P=0,04). O HLA-DRB1*04 e o HLA-DRB1*07 não apresentam associação com a HAI e colangite autoimune no grupo de pacientes mais jovens.

CONCLUSÃO:

Este trabalho demonstra uma associação dos principais polimorfismos no gene HLA-DRB1 à HAI com ou sem colangite na população brasileira.

DESCRITORES: Hepatite autoimune; Colangite esclerosante; Cadeias HLA-DRB1; Polimorfismo genético

INTRODUCTION

Type 1 autoimmune hepatitis (AIH) is a rare chronic inflammatory liver disease associated with loss of immunological tolerance to self-antigens1-3. Histologically, AIH is characterized by interface chronic hepatitis in the absence of a known cause1. AIH is uncommon in children, occurring between 10 and 30 years old2. Clinically, the disease can vary from asymptomatic liver disease to hepatic failure2,3. AIH type 1 overlap syndrome with autoimmune cholangitis or overlap syndrome (OS) is more common among children and adolescents, reaching almost 30% to 50% of pediatric population3,4. OS is characterized by AIH with features of primary sclerosing cholangitis4.

The etiology of AIH is not entirely known. As a complex disorder, many genes may interact with environmental factors resulting in disease5-8. Susceptibility to AIH is partially determined by the presence of genes related to major histocompatibility complex II (MHC II), especially the allelic variants of DRB1 of human leukocyte antigen (HLA)8-11. Among white North American and Northern European individuals, AIH has been associated with HLA-DRB1*03 and HLA-DRB1*046,12-16. In children, HLA-DRB1*13 is related to susceptibility to AIH, to the prognosis and to the response to treatment17. Czaja et al. 2002 concluded that patients with HLA-DRB1*03 were younger at disease onset than patients with HLA-DRB1*04, and they also had worse response to treatment with corticosteroids17. Patients expressing HLA DRB1*04 were more often women, with a greater chance of exhibiting other autoimmune diseases and with good response to corticosteroids17,18.

Few studies have evaluated the frequencies of HLA-DRB1 gene polymorphisms in pediatric patients with AIH and their relationship with clinical features. In this regard, the purpose of this study was to describe frequencies of the polymorphisms in HLA gene and to investigate the association of these polymorphisms with AIH and with OS. All findings were compared to healthy age and sex-matched controls.

METHODS

Patients and controls

This cross-sectional study included 43 pediatric patients, being 25 with type 1 AIH and 18 with OS (type 1 AIH overlap syndrome with autoimmune cholangitis). Patients were followed at Hepatology Reference Center of our institution (Hospital das Clínicas, Universidade Federal de Minas Gerais - UFMG, Brazil) from January 1986 to January 2014. Diagnosis of AIH was established according to the criteria of the International Group for the Study of AIH, published in 1993 and revised in 1999 and 200819,20. Diagnosis of OS was based on the presence of abnormalities in biliary tract, including stenosis and/or dilatation of intra- and/or extra-hepatic ducts on magnetic resonance imaging (MRI) of the biliary tract. MRI was performed in patients with persistent elevation of gamma glutamyl transferase (GGT) and/or poor response to immunosuppressive treatment. Three pediatric hepatologists and two radiologists independently reviewed the biliary tract images. Liver histology was also assessed.

Control group included 57 healthy individuals; age and sex matched with patients and the samples were collected at National Institute of Molecular Medicine Center (UFMG, Brazil). Healthy status was determined through the subjects’ medical history and either a parental report or self-report to rule out the presence of personal and family history of autoimmune diseases.

The study was approved by the Ethics Committee of our institution under the protocol ETIC#0419.0.203.000-10. Research protocol did not interfere with treatment. The consent form was read and signed by parents, researchers and patients when appropriate.

Clinical and laboratory monitoring

Follow-up visits consisted of clinical and laboratory evaluations at each 1 to 6 months, according to disease onset, protocol for diagnosis and therapeutic approach. Other etiologies of liver diseases were investigated and properly excluded, including infectious hepatitis, Wilson’s disease and deficiency of alpha-1-antitrypsin. All patients performed liver biopsy for disease staging. Diagnosis of OS was confirmed with the MRI of hepatic biliary tract and histological findings. Clinical features at disease onset were described for patients with AIH and with OS.

DNA Extraction and PCR-SSP

After informed consent, patients and controls were submitted to an intravenous puncture to collect 10 mL of peripheral blood samples. Genomic DNA was extracted using a saline method from the total blood21. The DNA concentration was adjusted to 50 ng/uL subsequently. HLA-DRB1 locus was performed using polymerase chain reaction and sequence-specific primers (PCR-SSP), as described elsewhere22. The polymorphisms evaluated were HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, and HLA-DRB1*13. The alleles sequence specific primers were designed based on previously reported reference23 and confirmed from GenBank database. The primers were synthesized by Thermo Fisher Scientific, Invitrogen and Integrated DNA Technologies (IDT).

Every PCR reaction tube contains 50 ng genomic DNA to be tested, 0.5 U Taq polymerase, 0.5 mmol of dNTP, 1.5 mmol of MgCl2, 0.2 mmol SSP of each HLA-DRB1 alleles. Conditions for PCR: denaturation at 95°C for 1 min; annealing temperature was specific to each primer for 30 s; elongation at 72°C for 1 min, 35 cycles. PCR products were analyzed by electrophoresis (100V, 400 mA, 60 min) of 1% ethidium bromide agarose gel. Two polymorphisms (HLA DRB1* 04 and HLA- DRB1* 07) were visualized in silver-stained 6.5% polyacrylamide gel.

Statistical analyses

Statistical analysis was performed using the software SPSS (IBM, Chicago, IL). Variables were checked for Gaussian distribution and were expressed as means and standard deviation or medians and interquartile range, when appropriated. Categorical variables and percentages were compared with chi-square and Fisher’s exact test. Unpaired student t-test was used for comparisons of means between two groups, while Mann Whitney test compared medians of two groups. The chance to have AIH or OS based on the presence of polymorphisms was expressed as odds ratio (OR) and confidence intervals (CI). For all comparisons, the level of significance was set at P<0.05.

RESULTS

Clinical and demographic characteristics of patients and controls

A total of 100 individuals were investigated, being 43 patients with AIH or OS and 57 healthy controls. Patients were divided in two subgroups: AIH (n=25) and OS (n=18). Controls were age (15.86±6.34 years in patients versus 13.92±4.55 years in controls, P>0.05) and sex-matched with patients (65% of females among patients and the same percentage among controls, P>0.05) at the time of blood collection.

In regard to disease onset, no significant differences were detected when patients with AIH were compared to patients with OS (Table 1).

TABLE 1 Comparison of the main clinical features of patients with AIH and with OS. 

Autoimmune Overlap P
hepatitis syndrome value
(n=25) (n=18)
Mean of age at disease onset (years) 9.1 (±3.1) 8.52 (±3.3) 0.61
Gender (females n) 19 (76%) 10 (55.5 %) 0.15
Disease presentation, number of patients (percentage)
Liver failure 2 (8%) 3 (16.7%) 0.38
Acute onset 8 (32%) 2 (11.1%) 0.10
Chronic liver disease 12 (48%) 9 (50%) 0.89
Asymptomatic 3 (12%) 4 (22.2%) 0.37

Laboratory data from patients with type 1 AIH and with OS (type 1 AIH overlap syndrome with autoimmune cholangitis)

Comparison of the laboratory features between patients group are presented in Table 2 and there was no statically difference in the parameters evaluated (Table 2). We have autoantibodies positivity and laboratory measures data from 24 patients with type 1 AIH (one patient evolved to death) and 18 patients with OS.

TABLE 2 Comparison of the laboratory features between two groups of patients with AIH and AIH in association with autoimmune cholangitis (overlap syndrome). 

AIH (n=24 AIH associated with P value*
patients) autoimmune cholangitis (n=18 patients)
Autoantibodies positivity
ASMA 19 (79%) 15 (83%)
ANA 16 (67%) 12 (66%)
ANTILKM1 01 (4%) 0 (0%)
Laboratory measures (median and Std Dev.)
GGT 123.2 (±72.84) 182.7 (±122.00) 0.06
Albumin 3.52 (±0.62) g/dL 3.59 (±0.56) g/dL 0.72
Gammaglobulinemia 2.70 (±1.07) g/dL 3.68 (±1.48) g/dL 0.05
Prothrombin activity 60.29 (±19.24) % 62.72 (±20.60) % 0.69

AIH: autoimmune hepatitis. *Statistical significance.

Frequencies of polymorphisms

Table 3 and Table 4 presents the frequencies of polymorphisms of HLA-DRB1* gene in patients with AIH, OS and in control group. The polymorphism of HLA-DRB1*13 had higher frequency in AIH group than in overlap syndrome or control group. The frequencies of other polymorphisms of HLA-DRB1* gene (03, 04 and 07) were similar in patients with AIH, with OS and in control group.

TABLE 3 Frequency of polymorphisms of the HLA-DRB1* gene in AIH, OS and control groups. 

Groups Polymorphism of HLA-DRB1*
0301 0401 %(n) 0701 %(n) 1301 %(n)
AIH 12% (12) 5% (5) 1% (1) 19% (19)
Overlap syndrome 9% (9) 1% (1) 2% (2) 8% (8)
Control 7% (7) 4% (4) 11% (11) 8% (8)
Total 28% (28) 10% (10) 14% (14) 35% (35)

AIH: autoimmune hepatitis.

TABLE 4 Numbers and percentages described are related to 100 individuals (43 patients and 57 controls).  

Polymorphism of AIH OS Controls
HLA-DRB1, n (%)100 patients patients
0301 12 9 7
0401 5 1 4
0701 1 2 11
1301 19 8 8

AIH: autoimmune hepatitis. OS: overlap syndrome.

Polymorphisms’ analyses

When patients with AIH and OS were put together and compared to controls, it was found that HLA-DRB1*13 polymorphisms were approximately 7-times more frequent in patients group than in controls (OR=7.2, CI 2.83 to 19.94; P<0.001). Regarding HLA-DRB1* 03 polymorphism, the frequency in the entire group of patients (AIH plus OS) was 3.5-fold higher (OR=3.57, CI 1.31 to 9.73; P<0.01) if compared to controls. For the HLA-DRB1*07 and HLA-DRB1*04 polymorphisms, there was no statistical difference (OR=0.21, P=0.057) between patients and controls (Table 5).

TABLE 5 Logistic regression of polymorphisms HLA-DRB1. 

SNP B (SE) OR (95% CI) P
DRB1*03 1.27 (0.51) 3.57 (1.31-9.73) 0.013
DRB1*07 -1.56 (0.82) 0.21 (0.04-1.04) 0.057
DRB1*13 2.02 (0.50) 7.52 (2.83-19.94) 0.0001

SNP: single nucleotide polymorphism. R2=0.95 (Hosmer and Lemeshow), 0.28 (Cox and Snell), 0.37 (Nagelkerke), X2 (3) of Model =32.33, DRB1*04 - variable removed in step 3 of 5. B: beta value; SE: standard error; OR: odds ratio; CI: confidence interval.

We also compared the frequency of polymorphisms between the groups of patients presenting AIH and AIH associated with autoimmune cholangitis. In this analysis, only HLA-DRB1*13 presented a significantly increased chance of about 4-fold of increase in the autoimmune cholangitis (OR=3.96, P=0.04) (Table 6).

TABLE 6 Logistic regression from polymorphisms HLA-DRB1 on groups AIH and overlap syndrome. 

SNP B (SE) OR (95% CI) P
DRB1*13 1.38 (0.67) 3.96 (1.07-14.61) 0.04

SNP: single nucleotide polymorphism. R2=0.09 (Cox and Snell), 0.13 (Nagelkerke), X2 (1) of the Model =4.481, DRB1*03 - variable removed in step 5 of 7, DRB1*04 - variable removed in step 4 of 7, DRB1*07 - variable removed in step 2 of 7. B: beta value; SE: standard error; OR: odds ratio; CI: confidence interval.

DISCUSSION

Our results demonstrate the HLA-DBR1*13 and HLA-DBR1*03 alleles were more frequent in the case than in comparison group. These results are in agreement with other studies18,24,25, since HLA-DBR1*13 polymorphism was more frequent in youngers. In the Fainboim 2001 study, the HLA-DBR1*13 polymorphism predisposes to a persistent hepatitis A virus infection26. Czaja et al. 2002 incorporated the HLA-DBR1*13 alleles into the hypotheses of disease pathogenesis18. One of the main proposed mechanisms for the susceptibility determined by the HLA molecule to autoimmune diseases is the selective attachment of peptides to MHC molecules associated with diseases. Based on this, some groups have investigated common amino acids following the HLA molecules translated by the major alleles involved in AIH susceptibility. Analysis of the amino acid sequence transcribed by the three alleles present in caucasian AIH patients, which are HLA-DBR1*03, HLA-DBR1*01, and HLA-DBR1*04, have an increased frequency of lysine at position 71 of the third region of the DRβ chain8,24. This functional change could explain the susceptibility to disease in patients presenting with the HLA-DBR1*0301 polymorphism. As HLA DRB1*13 was associated with the highest relative chance for the disease, it is possible that a lysine at position 71 is not implicated in susceptibility to AIH-1 in the studied population24. On the other hand, the DRB1*13 and DRB1*03 alleles display high sequence homology to other relevant peptide binding sites. In fact, the gene encoding the DRB1*03 specificity evolved from DRB1*13 by gene conversion in the HLA-DRB1*13 / DRB3 haplotype. Thus, as both molecules are present in 91% of Brazilian AIH-1 patients, other common amino acid residues in the HLA-1 molecule site are shared24 in some groups of South American children25. In Caucasians, the association also presented similar intriguing findings with the same HLA genes with autoimmune cholangitis in adulthood onset27,28.

The HLA-DRB1*07 and HLA-DRB1*04 polymorphisms did not demonstrate statistical significance for association with AIH. Other studies have shown the HLA-DRB1*07 polymorphism acting as a protector against the disease, because it has a reduced frequency in the studied patients13. However, the HLA-DBR1*04 polymorphism in previous research in European Caucasian population showed a 4 to 5-fold higher chance to present AIH (RR=3.9 and 4.97)15,16. In this study, the HLA-DRB1*04 and HLA- DRB1*07 does not show any statistical significance.

Other research in the Brazilian population did not find an association of HLA-DRB1*04 polymorphism in patients with AIH24. Asian populations, like the Japanese, seems to have a higher frequency of all HLA-DRB1*04 alleles in adult-onset with AIH-1. The HLA-DRB1*0405 allele was also investigated in adults with AIH-1 in Argentina and with the acute disease in Mexico18,29,30. There was no change in the frequency of HLA-DRB1*04 in the population studied, or any Brazilian population24.

The patient’s group was subdivided into groups according to the way the disease presented clinically to investigate predictive factors of the clinical presentation. Then, these patients were evaluated about polymorphisms to investigate if they would be able to predict the presence or absence of cirrhosis in patients with AIH or AIH associated with overlap syndrome. None predictive factor was statistically significant for the groups of patients studied (P>0.05). When comparing the polymorphisms between the AIH and autoimmune cholangitis groups, the only one able to predict a 3-fold increased chance of the presence of an overlap syndrome was the polymorphism HLA-DRB1*13 (OR=3.96, P<0.05).

This study is a case series composed of 43 patients, subdivided into AIH and AIH associated with overlap syndrome. This disease is a very rare disease, so the sample size is a real limitation for any assumption. In regression, the determination of sample size will depend on the extent of the effect being attempted and how much power is needed for this effect. As a general rule, it is possible to have 10 or 15 data for each model predictor28. For the first experiments, which included patients and controls, our sample size was 100 individuals for two predictors; these predictors were the presence or absence of the disease. These analyses were statistically significant. Secondary, the sample size was reduced to 43, to investigate whether the polymorphisms would predict AIH cirrhosis and evolution to autoimmune cholangitis. It was possible to observe statistical significance. However, in the last analysis to have an ability to predict outcomes having a reduced sample size (n=43), using four variables (DRB1*04, DRB1*07, DRB1*03 and DRB1*13) we have sample constraints. In view of the presented, larger sample is necessary to evaluate the ability of these polymorphisms to predict the degree of aggravation of the disease and the presence or absence of cirrhosis in the population with the evaluated characteristics.

CONCLUSION

AIH and its association with cholangitis autoimmune are a challenging condition for the patient and the scientific community. As a rare disease, the main limitation of this study was the reduced number of patients evaluated with AIH and this condition associated with cholangitis.

However, this work contributed to the investigation of the main polymorphisms in the HLA-DRB1 gene associated with AIH with or without cholangitis in young population. It has been shown that children with HLA-DRB1*03 and HLA-DRB1*13 polymorphisms are more likely to be affected with AIH, and children with HLA-DRB1*13 polymorphism are still likely to develop the association with autoimmune cholangitis.

ACKNOWLEDGEMENTS

Grants from FAPEMIG, CNPq and CAPES who support the research in Brazil.

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Disclosure of funding: no funding received

Received: January 08, 2019; Accepted: March 19, 2019

Corresponding author: Débora Marques de Miranda. E-mail: debora.m.miranda@gmail.com, deborammiranda@ufmg.br

Authors’contribution: Nunes MEG: acquisition, analysis and interpretation of data; statistics analysis; manuscript preparation. Rosa DV: scientific, conception and design of the study; manuscript preparation. Fagundes EDT, Ferreira AR: conception and design of the study, sample collection, paper revision. Miranda DM: scientific, intellectual, conception and design of the study; manuscript preparation, critical revision. Ferri Liu PM: scientific, intellectual, conception and design of the study; critical revision.

Declared conflict of interest of all authors: none

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