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Biomedical Research

No Association between HLA-DQB1 Genotypes with Nasopharyngeal Carcinoma in Batak Ethnic Groups in Indonesia

Author(s): Delfitri M.

Vol. 22, No. 2 (2011-04 - 2011-06)

Delfitri M.

Department of Ear Nose and Throat, Medical Faculty of Universitas Sumatera Utara, Nort Sumatera, Indonesia


Human Leucocyte Antigen (HLA) has been postulated to have an important role in the de-velopment of nasopharyneal carcinoma. To investigate the relationship between Human Leucocyte Antigen-DQB1 (HLA-DQB1) alleles and nasopharyneal carcinoma among Batak ethnic people, a case-control study was performed using samples consisting 55 cases and 104 controls. Deoxyribo Nucleic Acid (DNA) isolation was conducted at the Terpadu Laboratory of Universitas Sumatera Utara, while gene allele identification was carried out using poly-merase chain reaction sequence specific oligonucleotide reverse dot blot method at The De-partment of Immunohematology and Blood Transfusion, Leiden University Medical Center. The majority of nasopharyneal carcinoma cases were males (60%) and of 50-59 years old (29.09%). Histopathologically, the majority was of World Health Organization (WHO) type 3 (54.54%), followed by World Health Organization (WHO) type 1 (29.10 %) and World Health Organization (WHO) type 2 (16.36 %). Most of them are from stage III (67.28 %), followed by stage IV (25.45 %) and stage II (7.27 %). The result shows that there is no asso-ciation between Human Leucocyte Antigen-DQB1 (HLA-DQB1) gene in the case and the control samples (p>0.05). In conclusion, this study demonstrates that Human Leucocyte Antigen-DQB1 (HLA-DQB1) allele gene has no role in the development of nasopharyneal carcinoma among the Batak ethnic groups.

Keywords: Nasopharyngeal carcinoma, human leucocyte antigen, polymerase chain reaction sequence specific oligonu-cleotide reverse dot blot
Accepted December 29 2010


The exact etiology of nasopharyngeal carcinoma (NPC) to this date is still unknown. Nevertheless, there are some evidences to suggest that intrinsic factors such as Human leucocyte antigen (HLA), and extrinsic factors such as Epstein-Barr virus (EBV), nitrosamine, and environ-mental factors might be responsible in the etiology of NPC [1,2].

NPC among Chinese in South China is 100 times more prevalent than among the Caucasians [1]. The prevalence of NPC in the Chinese from the province of Guangdong is 39.84 per 100,000 people, while the populations in Japan, India and among the Caucasian were reported to be the lowest [3,4,5,6]. In Indonesia, its prevalence is 3.9 per 100,000 people [7]. The NPC in the Adam Malik Hospital in Medan, Sumatera Utara has predominantly been found within five ethnic groups. Among those ethnic groups, Batak has the highest incidence which is 46.7%.[8]. The risk for contracting the disease is increased in the pres-ence of family history of NPC. Data have shown that clusters of NPC have been reportedly found among the patients’s offspring [9]. The risk for NPC is still high among Chinese people who reside in places with low incidence of NPC [1]. Differ-ences in the frequency of NPC occurrence between tribes, increased risk for families with history of NPC, and high incidence among Chinese immigrants point to the role of genetic factors in the development of NPC. Among stud-ied genes, human leukocyte antigen (HLA) is believed as the most important factor in NPC [9,10].

HLA gene is a gene complex located within 4000 kilo-bases at short arm of chromosome number 6 and has a polymorphic character. This gene expresses HLA molecule on cell surface, which has an active role in the im-mune system [11].

One of the theoretical basis supporting relationship be-tween HLA gene and a disease is the immune response gene theory. In this theory, when antigen such as a virus infects the body, it undergoes many phases, which ends in the elimination of the antigen itself. HLA serves as a ge-netic marker in tissues and influences immune response, in this way it is important as a trigger of immune re-sponses [12].

HLA gene is inherited as heterozygote and co-dominant. Population group with characteristic HLA, will be at risk for certain diseases [13]. HLA gene or molecule distribu-tion in population has character and similarities that match with main pattern of HLA system group. Several diseases was suspected to be related with HLA, which means patients with the certain diseases are frequently detected to have certain alleles of HLA gene [14].

Several researches have found association between HLA and NPC, in which HLA can be susceptible or protective. In Taiwan it was shown that persons who have detected HLA-DQB1٭0201 gene are susceptible for NPC [15]. In Tunisia detection of alleles HLA-DRB1٭03 and HLA-DRB1٭15 genes shows susceptibility gene NPC [16]. Alleles HLA-DRB1٭1501 gene in Caucasians in United States has found as protected gene for NPC [17].

The research had been already found that association of HLA and other disease such as HLA-DR4 associated with rheumatoid arthritis [18]. Cervical cancer had been al-ready found had an association with HLA DQB1 ٭0503 [19].

Several research have proven that Epstein-Barr virus (EBV) has strong relationship and consistency as the causative factor for NPC [20,21]. There are evidences that EBV infection has strong relationship with personal cellu-lar immune power, as well as relationship with immuno-geneticity [22]. In this process, one of the factors that have a role is the HLA gene that works as a regulator in the immune process, and in the individual genetic marker [23]. HLA class II such as HLA-DQB1 gene is important for recognition process in immune response, and for the most determining success of immune system against EBV [24]. In latent infection, EBV produces several proteins such as membrane cell glycoprotein and nuclear cell anti-gen that play a role in the malignant transformation [25].

Indonesian people consist of several ethnic groups and are therefore influenced by several previous ethnic migration consequences. Risk of contracting to diseases varies in accordance to ethnic groups [26]. It is therefore important to know general image of alleles HLA gene of Indonesian NPC’s patients.

Aim of the Study

The aim of this study is to find HLA-DQB1 gene allele that is associated with the susceptibility of NPC develop-ment among Batak people. It is hoped that the research enefit is to find certain alleles HLA-DQB1 gene for giv ing preventive treatment and for discovering appropriate vaccines by using genetic engineering technique to pre-vent NPC’s among the Bataknese in the future.

Material and Methods

This explorative-observational study with case-control approach used the NPC cases among Batak people who sought treatment at Adam Malik, Pringadi, and Tembakau Deli hospitals located in Medan, North Sumatera, who fulfilled the inclusion criteria and the exclusion criteria. Inclusion criteria are all patient who are of Batak descen-dants with evidence of development of tumor(s) in the nasopharynx and later confirmed histopathologically as having carcinoma of the nasopharynx. The histopa-thological classification was performed based on World Health Organization (WHO) classification as WHO type 1, WHO type 2 and WHO type 3. The histopathology technique was done by hematoxylin eosin staining. Stag-ing of tumors was defined by American Joint Committee on Cancer staging method 2002. The exclusion criteria are all patients with NPC who showed Karnofsky per-formance status scale of test than <60% performance status, and or the samples are damage. We used diagnosis criteria for NPC in the study, when the histopathological tissue samples show evidence of derangement of the cel-lular lining that become dispolarized and the nuclear chromatin that become coarse, the nucleoli are prominent and the nucleo-cystoplasmic ratio is increased. The basal membrane can be intac in carcinoma in-situ or infiltrate through in the invasive type. The Batak tribes involved are Batak Toba, Mandailing, Karo, Dairi, Simalungun, Angkola, and Nias Control group comprised of Batak people who visited the above-mentioned hospitals, but with no cases of NPC, no relationship with case group, and no history of NPC in their families. The study was performed by unpaired case-control design, using α = 0.05 and β = 0.1. Based on Li et al (1995), minimal sam-ple size for each group was determined to be 45 [23]. The study sample is enrolled using consecutive sampling method.

Venous blood samples were collected from 55 NPC cases and 104 controls, and isolated at the Terpadu Laboratory of the Universitas Sumatera Utara. HLA-DQB gene al-leles were identified using reverse dot blot PCR-SSO at the Department of Immunohematology and Blood Trans-fusion, Leiden University Medical Center in the Nether-lands. Gene allele frequencies were determined based on the number of appearance from every sample.

NThe risk for NPC in certain HLA allel was calculated by odds ratio (OR) and for significance test we used Chi-Square test. Data analysis was carried out by SPSS 12.0 for windows program.


From this study we collected 55 case samples and 104 control samples.

Table 1. Sample Characteristic

Characteristic Total %
Male 33 60
Female 22 40
Age (year)    
20-29 1 1,82
30-39 13 23,64
40-49 15 27,27
50-59 16 29,09
≥ 60 10 18,18

The tables are of patients gender and age charac-teristic

Staging distribution above based on American Joint Committee on Cancer staging method 2002 (AJCC) stag-ing method 2002

Figure 1. Histopathology distribution.

Fig 1

The figure showed histopathology distribution based on WHO classification

Figure 2. Staging distribution.

Fig 2

Table 1. Association allel of HLA-DQB1 gene between cases and control, risk for NPC develop each allel gene

Table 1b


This research collected 55 case samples and 104 control samples. There is difference in male to female ratio, male is 60% and female 40%, with ratio is of 3 : 2. This shows similarity to what was reported by Morales-Angulo (1999) which showed that male was more affected than female [27].

This study showed that the most affected age group of NPC’s patients is of 50 – 59 years (29.09%). The young-est patient is 21 years old and the oldest 77 years old, whilst the average is 48.8 year. Data from Catarbia showed that the highest incidence age group is 50-70 years old [27]. In Adam Malik Hospital it was found the most age group is 50-59 year old [28].

The research showed that the most frequent histopha-tologic type is WHO type 3 (54.54%), while WHO type 1 was detected 29.10% and WHO type 2 is 16.36%. NPC WHO type 3 is the most common NPC case that was found in South East Asia [27]. According to Krishna (2004), this type was detected 100% EBV [29]. NPC WHO type 1 was predominant among Caucasian such as in Europe [30], while in Japan WHO type 2 is the most common [5].

In this research, the late stage was found to be 92.73% while early stage was 7.27%. (Stage III is the most case are 67.28% and stage IV is 25.45%. Stage II just 7,27%, while no stage I was found). In Guangzhou, China, stage III is the most common (68%) and also no stage I was detected [31]. In Japan more over stage IV the most common case (72%) [5]. In South of China it was found more case in early stage (87%) [32]. It is quite often that NPC was diagnosed in late stage, this is due to the fact that the location of nasopharyngeal space is hidden be-hind the nasal cavity. Jiang (2005) diagnosed that it can be caused by lack of knowledge about the spread of tumor and wrong interpretation in pathologic investigation. Early symptom of NPC is not characteristic and is often ignored by patient [33].

Thirteen allele HLA-DQB1 gene was investigated, and by Chi-square test there was no significant association be-tween allele HLA-DQB1 gene and NPC (p>0.05). An individual from Bataknese who has allele HLA-DQB1 gene, have not risk of suffering from NPC. The conclu-sion is that allele HLA-DQB1 gene has no role as the cause of susceptibility NPC development in Bataknese.

Allel DQB1٭0502 gene was found more in the study group (20%) compared to control (10.58%) with OR 2.11 (CI 0.85 – 5.25), that is suspected as susceptibility allele for NPC, however it does not show statistical significance (p> 0,05). Likewise allele HLA-DQB1٭0501 gene was detected less in the study group (18.18%) compared to control (25%) with OR 0,67 (CI 0,3 – 1,51), which is assumed as allele for protective gene, nevertheless this finding does not show statistical significance (p> 0,05). In Taiwan it was found that allele HLA-DQB1٭0201 gene showed susceptibility for NPC with risk of 2,6 [15]. In South of China it was not found association between HLA-DQB1 and NPC [23].

Polymorphism of HLA molecule was defined by se-quence of amino acid that forms binding cleft of peptide, and make interaction with antigen peptide and receptor of T cell. There is evidence that showed that more diversity of amino acid sequence at binding cleft is very important as a functional basis and some research showed as a struc-ture, as well as in the clinic although it is still experimen-tal [34]. Analysis of binding of antigen peptide, indicates that HLA molecules which is coded by different allele have different binding characteristic. Any molecule that has characteristic difference in the binding may cause petide antigen lost from the HLA molecule binding, and this may lead to a difference in the influence for T cell stimulation. There is an indirect evidence to support the concept that susceptibility and protectiveness of allele binding and their presentations are different in the their peptide epitope [35].

Individual suffering from NPC has susceptible gene that is not able to present or fails to present in the EBV anti-gen or in the malignant tumor bearing ThCD4 cell, result-ing in the failure of the host immune response [14]. Tu-mor antigen will infiltrate to the other cells and stimulate dissemination of the cancer [33]. Allele that protects gene works well to present EBV antigen or tumor antigen to ThCD4 cell. The response to the immune process is good, and the EBV as well as tumor antigen can be eliminated by body immune system and malignant process fails to occur [36].

There are differences in the susceptibility and protective-ness of allele HLA-DQB1 gene in each demographic area and ethnic groups, and it was suspected that it is due to the difference in the binding and in the presentation of the molecular peptide epitope. On the other hand, other than those factors the difference might be caused by the differ-ence in the sampling procedure, the difference of HLA distribution in each population and by the environmental factors such as etiology of NPC in this area.


The study showed that there is no association between allele HLA-DQB1 gene and susceptibility of developing NPC in Bataknese


The author was so much indebted to Prof. Frans Claas, who give laboratory facility in Department of Immuno-hematology and Blood Transfusion, Leiden University Medical Center, the Netherlands.


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Correspondence to:
Delfitri M.

Department of Ear Nose and Throat
Medical Faculty of Universitas Sumatera Utara
Address: Taman Setia Budi Indah II Blok IV No. 96 Medan, North Sumatera, Indonesia

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