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Abstract
Objective
Since the molecular basis underlying ciliopathies such as Bardet-Biedl (BBS) or Alström
(ALMS) syndromes is not fully understood, we hypothesised that changes in pattern
of DNA methylation, due to its role in embryogenesis and differentiation, could be
a mechanism that explains the pathogenesis of these diseases.
Methods
CpG islands search was performed by Methyl Primer Express software v1.0 (Applied Biosystems)
in the promoter region of ALMS1, BBS1, BBS2, BBS6, BBS7, BBS9, BBS10 and BBS12 genes.
In order to quantify the degree of methylation, we carried out MS-qPCR using SYBR®
Select Master Mix (Applied Biosystems). Blood lymphocyte DNA samples from seven patients
with ALMS were selected.
Results
Regarding to bioinformatic analysis, all genes harboured at least one CpG island.
Some of them included one or more sequences compatible with x-box motifs, which can
be recognized by transcription factors of RFX family that are known to be involved
in the regulation of ciliary genes transcription.
We selected a CpG island in the ALMS1 gene containing 67 cytosine residues potentially
methylated for performing MS-qPCR. A mean efficiency ranging from 90 to 96% was reached
for each amplicon in which the CpG island was divided. Unfortunately, no methylation
was detected in the enrolled patients.
Conclusion
Although the results of this preliminary study were negative, limitations due to sample
size, sample type and experimental approach have to be taken into account. However,
we consider it worth exploring this mechanism in BBS and ALMS using different techniques
such as methylation arrays, which could provide more accurate data.
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Conference name:
Cilia 2014 - Second International Conference