Background/Purpose: Our focus has been on the potential role of the epigenetic processes,
mainly of the methylation, on the development of Beh�et syndrome (BS) using
family data. We have previously studied global methylation in a group of
concordant and disconcordant 8 BS twins (4 monozygotic, 4 dizygotic) along with
healthy controls twins and found no difference within or between groups (average
dC:5-mdC ratio=0.032 sd=0.0006 for BS twins, and average dC:5-mdC ratio=0.030
sd=0.0001 for control twins). Further analysis on the role of HLA-B51 carrier
risk and its methylation mark using locus specific methylation analysis on
HLA-B region had indicated indicated increased methylation levels of BS twins
compared to healthy controls (p=0.0024)
(Ozkõlõnc M. et.al., Methylation Pattern of Twin Groups with Beh�et Syndrome.
International Congress- ASHG Annual Meeting 2012, San Francisco, California,
USA).

In this study, we wanted to take further our preliminary findings among
familial BS cases and more directly examine the influence of HLA-B51 frequency
and its methylation upon the presence of the clinical phenotype.

Methods: : About 850 BS patients seen consecutively at the Cerrahpasa Medical
Faculty, rheumatology outpatient clinic between 2013 and 2015 were asked if
they had an affected relative with BS. About 150 cases were familial and we
studied 15 index patients including their BS relatives (n=17) and their healthy
relatives (n=16).  All were ³ 20
years of age. Age-gender matched healthy controls were also included (n=25).
Peripheral blood samples were collected and genomic DNA was isolated from
leukocytes. HLA-B51 genotyping was performed by sequence-specific PCR and
random samples were confirmed with Sanger sequencing. HLA-B locus specific
methylation levels were analysed using Real-Time based OneStep qMethyl Kit. Chi-square
tests were performed to determine significance levels of genotyping results
among groups. Statistical analyses were performed using Graphpad Prism 6th
Version programme (GraphPad Software, La Jolla California USA).

Results: HLA-B51 carrier rate was significantly higher among index patients
(13/15),  affected relatives (13/17)
and healthy relatives (13/16) compared to healthy controls (8/25), (p<0.0001). As seen in the Table 1,
both index BS patients and their affected relatives had statistically higher
methylation levels for exon-1-intron-1-exon-2 region of HLA-B gene compared to
their healthy relatives and healthy controls (p=0.0044). Methylation
levels of index patients and affected relatives were similar. This was also
true for the methylation level of healthy relatives and healthy controls.

Conclusion: These findings point to a role of allele-specific methylation on BS
pathogenesis and deserve further scrutiny.

Table 1:Mean methylation levels and
mean age for four groups.