HLA B27 Subtype Distribution Among Patients with Ankylosing Spondylitis in Eastern Turkey

Abstract

Human leukocyte antigen (HLA) B27 has a strong association with ankylosing spondylitis (AS) and other spondyloarthropathies. More than 70 subtypes of HLA B27 have been described. The present study investigated B27 subtype distribution among B27-positive patients with AS classified according to the modified New York criteria and healthy controls. Sequence-specific primer polymerase chain reaction technique was used for B27 subtyping of 43 unrelated patients with AS and 39 volunteer bone marrow donors. Among patients with AS, the male-female ratio was 6.2 and the mean age was 30 years. No relationship was found between the B27 subtypes and clinical and laboratory findings in patients with AS (p>0.05). Similarly, the frequencies of B27 subtypes did not significantly differ between patients and controls. In this study, B*2746, B*2749, and B*2767 subtypes were detected for the first time. Among B27 subtypes, the most common B27 alleles found in the patients and the controls were B*2702 and B*2705. In addition, B*2702 subtype was found predominantly in both patients (48.8%) and controls (46.2%). In conclusion, in addition to commonly encountered B*2702 and B*2705 HLA subtypes, a B*2749 subtype in a patient with AS and B*2746 as well as B*2767 subtypes in controls were determined for the first time.

Introduction

Many rheumatic diseases result from the interaction of environmental and genetic factors. During the past 30 years, the definition of the association of B27 with ankylosing spondylitis (AS) has served as a milestone for research about the association of HLA and other genetic markers with spondyloarthropathies. However, the exact role of B27 in the etiology of AS is not completely understood. B27-positive individuals may not develop AS or other spondyloarthropathies, and 5-10% of patients with AS are negative for B27. There are few data in the literature about which triggers initiate spondylitis in the individuals with a genetic tendency (Akkoc and Khan 2008).

By now, more than 70 molecular subtypes of B27 have been described. Most of these subtypes differ from each other only by the properties of peptide-binding molecules of a few amino acids (IMGT/HLA Database 2011).

The distribution of B27 subtypes varies depending on ethnicity and geographic distributions (Khan and Ball 2002). Generally, HLA B*2705 is the most common subtype in all populations. HLA B*2702 exists in Asia and in certain countries of northern Africa, Europe, and the Mediterranean; it is reported to be the most common subtype among Caucasian and Arab populations (Khan et al., 2007). Several studies found that B*2705, B*2702, B*2704, and B*2707 are the subtypes predisposing to AS. B*2704 is the most commonly encountered subtype among Chinese and Japanese populations (Lin et al., 1996; Reveille 2006).

AS is a chronic progressive inflammatory disease that usually affects men (Bodur et al., 2010). On the basis of the data in the literature, it is not possible to determine whether HLA B27 subtypes in female patients help protect against this disease.

This study sought to determine B27 subtypes of B27-positive patients with AS and healthy controls. The local ethics committee approved this study.

Materials and Methods

Patient selection

This study included 43 patients with AS (37 men and 6 women) selected from referrals to the Rheumatology Policlinic of the Training and Research Hospital in Erzurum, eastern Turkey. The patients were diagnosed with and given medical treatment for AS according to the modified New York criteria, were unrelated, and were positive for the B27 allele.

Control population

The 39 control participants (27 men and 12 women) were selected among unrelated persons who had applied to our laboratory to be bone marrow donors and had previously been determined to be B27 positive. AS, reactive arthritis, psoriatic arthritis, and other auto-inflammatory or inflammatory immune disorders were ruled out in all controls.

HLA DNA typing

All patients and controls were typed for HLA B27 and allele frequencies at the Department of Medical Biology, Ataturk Faculty of Medicine. From all participants, 2 mL of peripheral blood was collected into test tubes that contained anticoagulant (EDTA). Genomic DNA was isolated by using an automated DNA isolation device (MagNA Pure LC DNA Isolation Kit I, Roche, Mannheim, Germany). B27 typing of all patients and controls was performed by using molecular methods. In patients, B27 positivity was assessed by using polymerase chain reaction (PCR) with low-resolution sequence-specific primers (One Lambda, Inc., Canoga Park, CA). Low-resolution HLA B typing of controls was performed by Luminex using PCR with sequence-specific oligonucleotide probes (Gene-Probe Transplant Diagnostic Inc., Stamford, CT).

Subtyping of B27 positive patients was performed by using PCR sequence-specific primers (One Lambda, Inc.). With the protocol used in this study, 57 distinct subtypes were analyzed. All subtypes were confirmed with One Lambda HLA fusion analyzing software.

Statistical analysis

Statistical analyses were done by using SPSS software, version 17.0 (SPSS Inc., Chicago, IL). The frequencies of B27 subgroups of patients with AS and healthy controls and clinical and laboratory measures of patients with AS were analyzed by using the chi-square method. Additionally, a Fisher exact test was used when necessary. The differences between the results obtained were considered significant at p<0.05.

Results

This study evaluated the clinical status and B27 subtypes of 43 B27-positive patients with AS. The male-female ratio among these patients was 6.2.

Table 1 summarizes clinical features of the patients. Clinical evaluation of the patients showed elevated serum C-reactive protein and erythrocyte sedimentation rate. Among the B27 subtypes, no relationship was found between patients' clinical features (Bath Ankylosing Spondylitis Functional Index, Bath Ankylosing Spondylitis Disease Activity Index, and radiologic evidence of sacroiliitis on x-ray defined in 1984 modified New York criteria) and laboratory values (erythrocyte sedimentation rate, C-reactive protein, white blood cell count, rheumatoid factor, and antistreptolysin O) (p>0.05).

Table 1.

Clinical and Demographic Features of Patients with Ankylosing Spondylitis

Characteristic	Data (n=43)
Age (y)	30±6.7
Men/women (n/n)	6/37 (ratio, 6.2)
BASDAI score (cm)	4.1±1.5
BASFI score (cm)	3.9±1.2
White blood cell count (10³/μL)	7.9±1.5
Antistreptolysin O (IU/mL)	114±111
ESR (mm/h)	23±20.8
C-reactive protein (mg/L)	12.5±12.9
Rheumatoid factor (IU/mL)	4.1±1.5
Radiologic sacroiliitis, n (%)	41 (95)

Data expressed with a plus/minus sign are the mean±standard deviation.

BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; ESR, erythrocyte sedimentation rate.

Table 2 shows B27 allele distribution among patients with AS and healthy controls. The frequencies of B27 subtypes of the patients and the controls did not differ (p>0.05). B*2702 and B*2705 were the most commonly encountered B27 alleles among the patients and the controls. Men and women did not differ for B27 subtype distribution (Table 3).

Table 2.

Distribution of HLA B27 Subtypes in Patients with Ankylosing Spondylitis and Controls

HLA B27 Subtype	Patients with AS (%)	Controls (%)	p value
B^*2702	48.8 (21)	46.2 (18)	0.808
B^*2704	2.3 (1)	0	1.000
B^*2705	39.5 (17)	41 (16)	0.891
B^*2707	0	5.1 (2)	1.000
B^*2712	4.7 (2)	0	1.000
B^*2717	2.3 (1)	0	1.000
B^*2746	0	2.6 (1)	1.000
B^*2749	2.3 (1)	0	1.000
B^*2767	0	5.1 (2)	1.000

Table 3.

Subtype and Sex Distribution in HLA B27-Positive Patients with Ankylosing Spondylitis

	Sex, n (%)
Subtype	Men	Women
B^*2702	18 (48.6)	3 (50)
B^*2704	1 (2.7)	0
B^*2705	14 (37.8)	3 (50)
B^*2712	2 (5.4)	0
B^*2717	1 (2.7)	0
B^*2749	1 (2.7)	0

B*2707 (n=2), B*2746 (n=1), and B*2767 (n=2) were identified only in healthy controls. B*2704 (n=1), B*2712 (n=2), B*2717 (n=1), and B*2749 (n=1) were identified only in patients with AS. B*2702 (n=18 for controls, n=21 for patients with AS) was the predominant subtype found in both patients and controls.

Discussion

Although B27 has a strong association with AS and spondyloarthropathies, the exact role of B27 in the etiopathogenesis of the disease is unknown (Khan and Ball 2002). In the Turkish population, the frequency of B27 was found to be 7-8% (Gul et al. 2002). That rate does not differ from that in other European populations. In our earlier study, B27 positivity was significantly higher in patients with AS (58%) than in healthy controls (4.3%) (p<0.05) (Dıyarbakır et al., 2011). B27 has 75 alleles with regard to nucleotide sequence diversity compared with 62 known subtypes at the protein level (Reveille 2006).

In the Turkish population, AS occurs more often in men than in women. In a study of 1381 patients with AS, prevalence of AS was 75.2% in men and 24.8% in women (Bodur et al., 2010). In our study, the male-female ratio was 6.2. The lower rate in women may be due to a milder disease course and lower disease index. An alternative explanation may be that women living in this region benefit much less from healthcare services than men do or appeal less often to the hospitals.

In our study, we did not aim to compare the HLA B27 positive patients to negative ones since it is widely accepted that B27-positive patients are likely to have more active disease and a worse disease course. Also, previous studies investigating B27 subtypes in patients with AS found no association between subtypes and clinical features or laboratory measures (Park et al., 2009; Wu et al., 2009).

In the Turkish population, the distribution of B27 subtypes shows a high rate of heterogeneity in comparison with other populations (Gunal et al., 2008). In our current study, 9 B27 alleles were identified. In European countries (e.g., Italy, Spain, and Greece), B*2702 and B*2705 are found in equal frequencies, whereas B*2702 is the predominant allele in northern Africa and in the Middle East (Gonzalez et al., 2002; Khan et al., 2007). In our study, B*2702 and B*2705 were the most common B27 alleles in patients and controls. Other investigators have reported that B*2705 was strongly correlated with AS in northern European countries and was found in 90% of patients (Baech et al., 1997). However, we noted no correlation between AS and B27 subtypes.

In this study, in contrast to other studies performed in Turkey, the predominant subtype was HLA B*2702 in both patients and controls. The B*2712 subtype found in some patients with AS was encountered earlier among Caucasian and Spanish people (Balas et al. 1998). In addition, B*2704 (B*270401) subtype, which was rarely found in the patients with AS, is more common in Asian populations (Brown et al., 1996). B*2707 subtype, seen in patients with AS in other studies but found in some controls in the present study, has been encountered before in Caucasian and Asian populations (Choo et al., 1991). In the current study, B*2749 was detected in patients with AS and B*2746 and B*2767 in the control group; to our knowledge, these subtypes have not been found in previous studies. It is argued that the high diversity of B27 subtypes originated from the geographic location of Turkey, which has served as a bridge connecting Europe and Asia for centuries.

In conclusion, in addition to commonly encountered B*2702 and B*2705 HLA subtypes, the B*2749 subtype in patients with AS and B*2746 and B*2767 subtypes in control participants were detected for the first time in this study.

Footnotes

Disclosure Statement

No competing financial interests exist.

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