Prevalence of HLA DQ 2,8 in children with celiac disease

Abstract

OBJECTIVE:

Celiac disease is a chronic disease that affect small bowel by making its villi become atrophic. Various environmental and genetic factors have been identify as inducing factors for celiac disease. Most of the patients has one of the HLA DQ forms. Although the prevalence of these genes are variable in different areas of the world, we do not have a comprehensive information about this issue in our region. Thus the aim of present study is to investigate the prevalence of HLA DQ typing of patients who visited Emam Reza Gastroenterology clinic of Shiraz(IRAN).

METHODS:

In this case-control study all under 18 years old children who were diagnosed with celiac disease and have visited Emam Reza gastroenterology clinic were investigated. The diagnosis of celiac disease was made by history, physical exam, serologic test, and histopathology of duodenal biopsy. Blood sample was taken and HLA typing performed using PCR method at Motahari clinic cytology laboratory. Also those people who neither them self nor their first degree relatives were not case of celiac disease and underwent HLA typing for other reason were identified as control group. The statistical analysis was done using SPSS 18 software. The $p$ value $<$ 0.05 was identified as statistically significant.

RESULTS:

A total of 139 patients with celiac disease and 146 normal children were studied. The mean age of the patient with celiac disease were 9.1 years old with standard deviation of 3.4 years old. 64% of the celiac patients were girls and 36% were boys. While this proportion was 54.4% for boy and 48.6% for girls in control group. The most common HLA in celiac patients group were HLA DQ2 and 8 but the most common ones in control group were HLA DQ 8 and 5. Failure to Thrive were the most common signs of the celiac patients with a prevalence of 60 children. Total IgA titer were normal in 98.6% of the patients and TTG IgA titer were positive in 93.5% of the patients. The most common co existing disease with the celiac disease were diabetes with a prevalence of 30 children (66.7%).

CONCLUSION:

present study reveals that the prevalence of the HLA DQ2 and 8 among patients with celiac disease is 72.6% and 53% in our normal population.

Keywords

Celiac disease HLA Iran

1. Introduction

Celiac disease is a chronic autoimmune disorder, which primarily affects the small intestine. The disease is characterized by atrophy of the small intestine mucosa. In general, celiac disease affects between 1 in 170 and 1 in 100 people. However, these rates vary, from as few as 1 in 300 to as many as 1 in 40, between different regions of the world [1]. The prevalence of the disease among the Iranian population is 0.72% (a 95% confidence interval (CI): 0.62%–0.98%) [2]. The disease affects the population of whites as well as women at a ratio of 3:1 more than men. Based on the 2010 rate, there are about 550000 symptomatic adults and about 240000 sick children: 85% of the symptomatic adults suffer from gastrointestinal disorders, 40% have anemia, and 30% have abnormal liver enzymes. The standardized medical costs, for patients with celiac symptoms, are estimated at around £ 4 billion (£ 287 million for children) over the next 10 years, with a delay between the onset of symptoms and diagnosis (on average, 6 years for adults, 2 years for children). A delay in diagnosis is expected to increase mortality; approximately 600000 celiac patients will die in the next 10 years, with an excess of 44.4% vs age- and sex-matched controls [3]. In affected individuals, there are Four defined components: Gluten, Tissue Transglutaminase (tTG), HLA-DQ2/8, and T cells. These components interact. In the gastrointestinal tract, gluten breaks down into relatively large fragments due to pepsin activity in the stomach. These fragments may be further degraded by enzymes in the small intestine, but due to the proline-rich nature of gluten, the fragments remain relatively large. Some of these with a little desire can be bound to HLA-DQ2 or HLA-DQ8-susceptible molecules and T cells, which are reactive for such DQpeptide complexes, although found in small quantities in patients with CD. However, the response of these T cells, probably in combination with the induction of intrinsic responses, can lead to tissue damage. This results in the release of the TG2 enzyme that can alter gluten peptides in an extracellular calcium-rich medium. This change is called Deamidation and involves the conversion of neutral glutamine amino acid to a negatively charged glutamic acid amino acid. As a result of this negative charge, the deamidated gluten peptides are more likely to bind to HLA-DQ2 or HLA-DQ8, because these HLA molecules prefer to bind to those peptides with one or two negatively charged residues [5]. In addition, a large number of gluten peptides can be altered in this way. It enhances the gluten-specific T cell response in lamina propria. This response is characterized by the secretion of specific proinflammatory cytokines such as INF- $\gamma$ that stimulate local inflammation. Importantly, these results illustrate the well-known fact that CD develops almost exclusively in individuals with the positive HLA-DQ2 or HLA-DQ8 type. The pathogenesis of CD stems from interactions between genetic factors (HLA and non-HLA genes), gluten, and other environmental stimuli that are still unclear. Among the genetic factors associated with CD, the strongest association is in the HLA class II region. More than 90% of patients carry one or two copies of HLA-DQ2.5 encoded by the DQA1*05 (alpha chain) and DQB1*02 (beta chain) genes. HLA-DQ2.2 is largely homologous to DQ2.5, but is associated with less risk due to reduced ability to bind peptides. The highest risk is related to the homozygosity of DQB1*02. Data on DQ2 negative patients indicate that they are almost invariably HLA-DQ8 positive (DQA1*0301 /DQB1*0302). Effective environmental factors include surgery, pregnancy, infection, and emotional stress [6]. Most people with celiac disease have one of two types of HLA-DQ protein. HLA-DQ is a part of the receptor system that presents the MHC class II antigen (also it is called human leukocyte antigen) and differentiates between innate and non-innate cells for the purposes of the immune system. The two subunits of the HLA-DQ protein are encoded by the HLA-DQA1 and HLA-DQB1 genes, which are located on the small arm of the chromosome 6. There are seven HLA-DQ variants (DQ2 and DQ4–DQ9). More than 95% of people with celiac disease have DQ2 or DQ8 isoforms, which is inherited in families. The reason that these genes increase the risk of celiac disease is that the receptors formed by these genes bind to gluten peptides more tightly than other forms of the antigen-presenting receptor. Thus, these forms of the receptor are more likely to activate T lymphocytes and initiate an immune response [7]. Most people with celiac disease carry a two-gene HLA-DQ2 haplotype, known as the DQ2.5 haplotype. This haplotype consists of two adjacent DQA1*0501 and DQB1*0201 alleles and encodes both DQ $\alpha$ 5 and DQ $\beta$ 2 subunits. In most people, this DQ2.5 isoform is encoded by one of the two copies of chromosomes 6 inherited from parents (DQ2.5cis). Most celiacs inherit only one copy of this DQ2.5 haplotype, although some people inherit it from both parents; the latter are particularly at risk for celiac disease, and are also more susceptible to severe complications [6]. Some people inherit DQ2.5 from one parent and an additional portion of the haplotype (DQB1*02 or DQA1*05) from the other parent, which increases the risk. In less common cases, some people inherit the DQA1*05 allele from one parent and the DQB1*02 allele from the other parent (DQ2.5trans) (it is called a trans-haplotype association), and these individuals are at similar risk for celiac disease as those with a single DQ2.5-bearing chromosome 6, but in this example, the disease it is not a familial one. Of the 6% of European celiacs who do not have DQ2.5 (cis or trans) or DQ8 (coded by the DQA1*03:DQB1*0302 haplotype), 4% have DQ2.2 isoform, and the remaining 2% lack DQ2 or DQ8. The abundance of these genes varies geographically. DQ2.5 is highly abundant in northern and western Europe and parts of Africa and India, but is not found in parts of the West Pacific rim. DQ8 has a wider global distribution than the DQ2.5, and is particularly common in Central and South America; up to 90% of people in the Amerindian population (mixed race American and Indian or Eskimo) carry DQ8, and so they may display the celiac phenotype [6]. Other genetic factors have been repeatedly reported in celiac disease; however, involvement in the disease has varied geographical recognition. Only the HLA-DQ loci show a consistent involvement over the global population. Many of the detected loci have been found in association with other autoimmune diseases. One locus, the LPP gene or the lipoma-preferred partner, is involved in the adhesion of the extracellular matrix to the cell surface, and a minor variant (SNP $=$ rs1464510) increases the risk of disease by approximately 30%. This gene is strongly associated with celiac disease in samples taken from a wide area of Europe and the United States [8]. Given the characteristics of celiac disease and its apparent strong heritability, it would normally be expected that the genotypes would undergo negative selection and to be absent in societies where agriculture has been practiced the longest. (Compared to a similar condition, lactose intolerance, which is greatly affected by negative selection, has decreased from about 100% in ancestral populations to less than 5% in some European countries). This expectation was first proposed by Simoons (1981). By now, however, it is apparent that this is not the case; on the contrary, there is an evidence of positive selection in celiac disease genotypes. It is suspected that some of them have some benefits by providing protection against bacterial infections [9]. The aim of this study was to determine the frequency of HLA DQ 2.8 in children with celiac disease referred to the Pediatric Gastroenterology Clinic of Imam Reza Clinic.

Table 1
The comparison of age, sex, allele and HLA of the children with celiac with healthy controlgroup

Variable	$P$ value	Control		Celiac
		SD	Mean	SD	Mean
Age, y	$<$ 0.001	13.2	32.2	3.4	9.1
		Percentage		Percentage	N
Gender
Male	0.012	54.4	75	36	50
Female		48.6	71	64	89
HLA
DQ2	$<$ 0.001	25	73	53.20	148
DQ3		0	0	0	0
DQ4		0.3	1	0.35	1
DQ5		33.5	98	12.23	34
DQ6		12.6	37	7.91	22
DQ7		0.6	2	4.3	12
DQ8		27.7	81	21.9	61
Allele
02	$<$ 0.001	25	73	53.2	148
03		27.7	81	21.9	61
03-01		0.6	2	4.3	12
04		0.3	1	0.35	1
05		33.5	98	12.23	34
06		12.6	37	7.91	22

2. Methods

All children under the age of 18 who were referred to the Pediatric Gastroenterology Clinic of Imam Reza Clinic in Shiraz during the years 2011–2017 and diagnosed with celiac disease, were studied as the case group, and patients who had neither celiac disease themselves nor their families, and for whom HLA was checked for other reasons, were included as the control group .Due to the lack of similar research in this field, the sample size was estimated to be at least 15 individuals in each group. In this cross-sectional study, all children under the age of 18 who were referred to the Pediatric Gastroenterology Clinic of Imam Reza Clinic in Shiraz during the years 2011–2017 and diagnosed with celiac disease, were selected. The diagnosis of celiac disease in these children is based on the history, clinical examination, serologic test using TTG-IgA and histopathological examination of duodenal biopsy specimens. Blood samples of all the patients were sent to the cytogenetic laboratory of Shahid Motahari Clinic for a review of HLA DQ 2.8, and HLA typing was performed by the polymerase chain reaction (PCR). A group of patients who had neither celiac disease themselves nor their families, and for whom HLA was checked for other reasons, were studied as the control group. Statistical indices such as median, mean and standard deviation were used to express the results. Chi-square test was used to compare age, sex, allele, and HLA between the case and control groups. The results were analyzed by SPSS 18 software. $P$ values less than 0.05 were considered significant. Written informed consent was obtained from all the patients in the study.

3. Results

The study included 139 patients with celiac disease and 146 subjects in the control group. The mean age of the patients with celiac disease was 9.1 years and its standard deviation was 3.4 years. These values in the control group were 32.2 and 13.2 years, respectively. This difference was statistically significant ( $p$ value $<$ 0.001). In terms of the sex ratio, the number of girls (64%) was more than boys (36%).

The most common HLAs among the patients with celiac disease were HLA-DQ2 and HLA-DQ8, while the most common HLAs in the control group were HLA-DQ8 and HLA-DQ5 ( $p$ value $<$ 0.001). Of the alleles on which HLA was located, the most common allele in the case group was allele 2 and in the control group it was allele 5 ( $p$ value $<$ 0.001).

Table 2
The anthropometric information of the children with celiac disease

Variable	SD	Mean
Height	19.4	128.0
Weight	11.2	26.7
BMI percentile	28.6	28.9

Table 3

Signs and symptoms of disease in the children with celiac disease

Sign and symptom	Number of patients	Percentage
Child developmental disorder	60	38.0
Short statur	38	24.1
Diabetes	31	19.6
Iron deficiency anemia	11	7.0
Abdominal pain	9	5.7
Chronic diarrhea	3	1.9
Osteopenia	2	1.3
Chronic constipation	1	0.6
Anorexia	1	0.6
Chronic fatigu	1	0.6
liver enzyme	1	0.6

As shown in Table 2, the mean height of the patients with celiac was 128.0 cm with a standard deviation of 19.4 cm. Their average weight was 26.7 kg with a standard deviation of 11.2 kg. Also, the mean percentile of BMI of the patients with celiac was 28.9 with a standard deviation of 28.6.

Failure to Thrive was the most common symptom in the children with celiac disease (60 cases, 38%). Other symptoms were short stature (38 cases, 24.1%) and diabetes (31 cases, 19.6%).

Table 4

Total immunoglobulin A titers and tissue transglutaminase in the children with celiac disease

Percentage	N	Variable
		Total IgA Tite
98.6	13	Normal
1.4	2	Deficiency
		IgA TTG Tite
93.5	13	Positive
6.5	9	Negative

As shown in Table 4, total immunoglobulin A titers were normal in 98.6% of the children with celiac disease, and only in two cases, there was a deficiency. Tissue transglutaminase was also positive in 93.5% of the children with celiac disease, and negative in only 6.5% of them.

Histopathological findings of samples taken from children with celiac disease indicated that the major histopathologic findings were Marsh 3B 56 cases, 40.6%) and Marsh 3A (50 cases, 36.2%).

In terms of comorbidities, the most common comorbidity among the patients with celiac disease was diabetes, which was observed in 30 patients (66.7%). Other comorbidities observed included thyroid disease (14 cases 31.1%) and autoimmune hepatitis (1 case, 2.2%).

Table 5

Histopathological findings of samples taken from the children with celiac disease

Histopathological findings	Number of patients	Percentage
Marsh 1	1	0.7
Marsh 2A	2	1.4
Marsh 2B	7	5.1
Marsh 3A	50	36.2
Marsh 3B	56	40.6
Marsh 3C	22	15.9

4. Discussion and conclusion

The aim of the present study was to evaluate the HLA in patients with celiac disease referred to Shiraz Pediatric Gastroenterology Clinic. The incidence of celiac disease in different geographical locations is different. For example, a review study showed that the incidence of celiac disease in Iran is about 1 in 166 people. The incidence is higher in Turkey at around 1 in 87, while in places like India, it is about 1 in 500 to 1 in 20,000, probably due to the higher prevalence of gastrointestinal infectious diseases in the country [22]. In terms of the sex ratio of the patients with celiac disease, girls (64%) were more than boys (36%). In terms of HLA, the most common HLAs among the patients with celiac disease were HLA-DQ2 and HLA-DQ8, while the most common HLAs in the control group were HLA-DQ8 and HLA-DQ5.

Table 6

The comorbidities in the children with celiac disease

Variable	Number of patients	Percentage
Diabetes	30	66.7
Thyroid diseas	14	31.1
Autoimmune hepatitis	1	2.2

In summary, HLA DQ 2/8 were found in 72.6% of the patients with celiac disease and 53% of the control group. The frequency of HLA DQ 2/8 in the case group was lower than in the previous studies. For example, in a study by Cecilio et al., it was showed that the presence of these two HLAs was 98.4% in the patients with celiac disease. But the percentage of HLA observed in the normal subjects was similar to ours in this study and was 55.4% [23]. However, in their study, they also examined the HLA of the patients’ families, which unfortunately was not addressed in our study, and showed that its frequency was 89.6% for them. Also, a similar internal study that examined HLA in the patients with celiac disease in the Iranian population also showed a 97% prevalence of the HLAs in the patients and a 58% prevalence among the normal subjects [24]. In another study by Di Giacomo, the frequency of HLA-DQ2 and HLA-DQ8 among people with gastrointestinal problems who did not have celiac disease was examined. The results of this study showed a higher incidence of these HLAs among patients with gastrointestinal and functional diseases of the upper gastrointestinal tract and a lower incidence of these HLAs among patients with irritable bowel syndrome and inflammatory bowel disease [25]. Failure to Thrive was the most common symptom in the children with celiac disease (60 cases, 38%). Other symptoms were short stature (38 cases, 24.1%) and diabetes (31 cases, 19.6%). In other studies, diarrhea is the most common symptom of celiac disease. According to other studies, it seems that certain functional diseases such as irritable bowel syndrome can influence the prevalence of HLA, which suggests that this should be considered in the control group. Typically, case-control studies try to match the case and control individuals in terms of age and sex as much as possible. Although in our study, there was a significant difference between the two groups, but this could not affect the results of our study because the HLA and allele of patients are not related to the age and sex of them, so the two variables (Age and Sex) are not considered as confounding factors. Ethnic and genetic differences in different geographic regions can affect the frequency and genetic characteristics of celiac disease. Although there have been some studies on the prevalence of celiac disease in the south of Iran, few studies have been conducted on genetic studies of celiac disease in these areas [26]. The results of this study show that the frequency of HLA DQ2/8 is higher in the celiac patients in our area than in the normal population, but it is lower than in celiac patients in other geographical areas.

References

Green

P.H.

and Cellier

, Celiac disease, New England Journal of Medicine 357(17) (2007), 1731–43.

Ahadi

Shafiee

Razmandeh

Keshtkar

Sani

and Azemati

, Prevalence of celiac disease among the Iranian population: A systematic review and meta-analysis of observational studies, Turk J Gastroenterol 27(2) (2016), 122–8.

Malekzadeh

Sachdev

and All

, Coeliac disease in developing countries: middle East, India and North Africa, Best Practice & Research Clinical Gastroenterology 19(3) (2005), 351–8.

Marsh

, Coeliac disease in developing countries: Middle East, India and North Africa, Best Practice & Research Clinical Gastroenterology 19(3) (2005), 351–8.

Kupfer

and Jabri

, Celiac disease pathophysiology, Gastrointestinal Endoscopy Clinics of North America 22(4) (2012).

Barker

and Liu

, Celiac disease: pathophysiology, clinical manifestations, and associated autoimmune conditions, Advances in Pediatrics 55(1) (2008), 349–65.

Koning

, Pathophysiology of celiac disease, Journal of Pediatric Gastroenterology and Nutrition 59 (2014), S1–S4.

Parzanese

Qehajaj

Patrinicola

Aralica

Chiriva-Internati

and Stifer

, Celiac disease: From pathophysiology to treatment, World Journal of Gastrointestinal Pathophysiology 8(2) (2017), 27.

Dieli-Crimi

énit

M.C.

and Núñez

, The genetics of celiac disease: A comprehensive review of clinical implications, Journal of Autoimmunity 64 (2015), 26–41.

10.

Byrne

and Feighery

C.F.

, Celiac disease: Diagnosis, Celiac Disease: Springe, 2015, pp. 15–22.

11.

Fasano

and Catassi

, Current approaches to diagnosis and treatment of celiac disease: an evolving spectrum, Gastroenterology 120(3) (2001), 636–51.

12.

Fasano

and Catassi

, Celiac disease, New England Journal of Medicine 367(25) (2012), 2419–26.

13.

Abdulkarim

Burgart

See

and Murray

, Etiology of nonresponsive celiac disease: results of a systematic approach, The American Journal of Gastroenterology 97(8) (2002), 2016–21.

14.

Tack

Verbeek

Schreurs

and Mulder

, The spectrum of celiac disease: epidemiology, clinical aspects and treatment, Nature Reviews Gastroenterology and Hepatology 7(4) (2010), 204.

15.

Gujral

Freeman

and Thomson

, Celiac disease: prevalence, diagnosis, pathogenesis and treatment, World Journal of Gastroenterology: WJG 18(42) (2012), 6036.

16.

Anderson

Henry

Taylor

Duncan

Danoy

and Costa

, A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways, BMC Medicine 11(1) (2013), 188.

17.

Houshiyar

Fouladi

Ghorbani

Mohammadi

and AlimohammadiAsl

, Prevalence of Human Leukocyte Antigen (HLA DQ2 and DQ8) in celiac disease in patients with irritable bowel syndrome, Journal of Ardabil University of Medical Sciences 15(3) (2015), 277–82.

18.

Biagi

Bianchi

Vattiato

Marchese

Trotta

and Badulli

, Influence of HLA-DQ2 and DQ8 on severity in celiac disease, Journal of Clinical Gastroenterology 46(1) (2012), 46–50.

19.

Barada

Bitar

Mokadem

Hashash

and Green

, Celiac disease in Middle Eastern and North African countries: A new burden? World Journal of Gastroenterology: WJG 16(12) (2010), 1449.

20.

Gastro-Antunes

Crovella

Branão

Guimaraes

Motta

and Silva

, Fequency distribution of HLA DQ2 and DQ8 in celiac patients and first-degree relatives in Recife, northeastern Brazil, Clinics 66(2) (2011), 227–31.

21.

De Silvestri

Capittinim

Poddighe

Valsecchi

Marseglia

and Tagliacarne

, HLA-DQ genetics in children with celiac disease: A meta-analysis suggesting a two-step genetic screening procedure starting with HLA-DQβ chains, Pediatric Research 83(3) (2018), 564.

22.

Nejad

Rostami

Emami

Zali

and Malekzadeh

, Epidemiology of celiac disease in Iran: A review, Middle East Journal of Digestive Diseases 3(1) (2011), 5.

23.

Cecilio

and Bonatto

, The prevalence of HLA DQ2 and DQ8 in patients with celiac disease, in family and in general population, ABCD Arquivos Brasileiros de Cirurgia Digestiva (ão Paulo) 28(3) (2015), 183–5.

24.

Rostami-Nejad

Romanos

Rostami

Ganji

Ehsani-Ardakani

and Bakhshipour

, Allele and haplotype frequencies for HLA-DQ in Iranian celiac disease patients, World Journal of Gastroenterology: WJG 20(20) (2014), 6302.

25.

DiGiacomo

Santonicola

Zingone

Troncone

Caria

and Borgheresi

, Human leukocyte antigen DQ2/8 prevalence in non-celiac patients with gastrointestinal diseases, World Journal of Gastroenterology: WJG 19(16) (2013), 2507.

26.

Saberi-Firouzi

Omrani

Nejabat

Mehrabani

and Khademolhesseini

, Prevalence of celiac disease in Shiraz, southern Iran, Saudi Journal of Gastroenterology: Official Journal of the Saudi Gastroenterology Association 14(3) (2008), 135.