Rubinstein-Taybi syndrome and keloids

Rubinstein-Taybi syndrome (RTS) is a multiple congenital anomalies-intellectual
disability syndrome, caused by mutations in CREBBP or its homologue EP300. One
of the complications in RTS is keloid formation occurring in 25-30% of
individuals. Keloids are proliferative fibrous growths resulting from excessive
tissue response to skin trauma. In RTS minimal or unrecognizable skin injuries
can cause massive keloid formation, thereby affecting quality of life in RTS
significantly due to their extensive itching.

The present proposal aims at detecting the pathogenesis of keloids in RTS by
studying 50 RTS individuals from 3 countries (Netherlands, Norway, UK) with
keloids clinically, and take 3 biopsies in 5 of them from normal skin, edge of
normal skin-keloid and keloid. Molecular studies (transcriptome analysis; HAT
activity) and cell biological functions (TGFß-signalling pathway; expression
studies) will be performed in frozen biopsies and cultured cells. Depending
results a topical medication will be chosen and prepared for a future trial.

Comprehensive literature review
a. RTS
b. Keloids
c. Keloids in RTS
d. Keloids in RTS and itching

Clinical studies
1:. All RTS individuals known to support groups in the Netherlands, Norway and
UK will be invited to participate through their national support groups (all
support groups have agreed to do so).
2. Parents/other legal representatives of RTS individuals with keloids can
indicate their willingness to participate at a dedicated e-mail address.
3. Check if mutation is known in RTS individual (studied in about 3/4 of
individuals); if not this will be offered for free during attendance of the
clinic. The clinical diagnosis in all members of the support groups of the
three countries is already confirmed by one of us (RCMH).
4. All will be invited to visit a clinic (one in each country). Families are
again informed in detail about the study, offered the opportunity to ask
questions, and asked to sign the consent form.
5. The clinical history will be obtained using 3 validated questionnaires
forwarded to participating families in advance (RTS; keloid; itching. See
Appendices 1-3). Results will be discussed during the clinic, and additional
questions asked in case of ambiguities.
6. Physical exam will be performed standardized using the Patient and Observer
Scar Assessment Scale (van de Kar 2005). Three dimensions of keloids will be
measured with a ruler. Standardized photographs will be taken of chest,
shoulders and back. 10cc venoud blood will be drawn for itching parameters.
7. Biopsies (2mm) will be performed in 5 RTS individuals from 3 sites: healthy
skin; active edge between keloid and healthy skin; keloid. The sites will be
discussed in advance between the physician and RTS individual/family. All
biopsies will be taken during anaesthesia needed for other reasons. Biopsies
will be stored using HAM F10 or similar medium and forwarded to the lab within
24 hours. Photographs will be taken just before and after biopsying of the
biopsy site. Follow-up will be offered to all 5 biopsied RTS individuals after
6 and 12 months, to check for new keloid formation which will be measured and
photographed.

Molecular studies
a. Part of the biopsy material will be used for fibroblast cultures using
standard protocols.
b. Other parts of the biopsies will be frozen to allow for mRNA and protein
expression studies at the particular part of the skin (normal; edge normal
skin-keloid; keloid).This will be done at T0, and after 1, 3 and 6 passages of
fibroblast cultures.
c. Expression studies of CREBBP and EP300 in the frozen biopsies using
quantitative PCR.
d. Transcriptome analysis in both cultured cells and frozen biopsies at mRNA
using deep sequencing (Illumina/Solexa). Data analysis, involving fold-change
calculations, standard statistical tests, including ANOVA analyses, and
clustering will be performed. Analysis of the function of differentially
regulated gene sets will use both Bioconductor and in-house software that
queries the Gene Ontology databases and assesses whether deregulated genes are
over-represented in these pathways. Data will also be compared with previously
established transcriptome sets in Growth factor stimulated T98G cells.
e. HAT function: we are presently testing which technique functions best to
test this in CREBBP: a filter-binding assay measuring the transfer of
radiolabeled acetate from acetyl-CoA to protein, or spectroscopic
enzyme-coupled assays linking the HAT reaction to reduction of NAD+ by pyruvate
or alpha-ketoglutarate dehydrogenase. Both techniques have specific
characteristics (Berndsen et al 2005) and in a presently running pre-study we
will determine which functions best for the present aims.
f. TGFß-signaling will be studied in cultured cells and frozen biopsies by
analysis of nuclear localization of P-SMAD2 by immuno-fluorescence on cells or
by immunohistochemistry. Also expression levels of TGFß, TFGBR (Alk5), P-Smad2
and expression of SMAD2 target genes (fibronectin, collagen type I, plasminogen
activator inhibitor 1 and matrix metalloproteinase-2) will be studied by
Western blotting and/or quantitative PCR.

Burden:
The patient will be asked to complete 3 questionnaires. A physical exam will
be performed and the POSAS (Patient and Observer Scar Assessment Scale) will be
completed. Three dimensions of keloids will be measured with a ruler.
Standardized photographs will be taken of chest, shoulders and back. 10cc of
peripheral blood will be taken for itching parameters. Biopsies (2 mm) will be
performed in 5 RTS individuals from 3 sites: healthy skin, active edge between
keloid and healthy skin, keloid. The sites will be discussed in advance between
the physician and RTS individual/ family. All biopsies will be taken during
anaesthesia needed for other reasons.

Risks:
The small biopsy wound taken from normal skin is unlikely to develop into a
keloid scar as it will be taken from an area not known to develop keloids (i.e.
lower limbs). The biopsies taken in the middle from the keloid will not cause
an increase of the keloid. Only the biopsy taken from the edge normal
skin-keloid might well cause an increase in size of the keloid of about 2mm. As
especially a biopsy from this site is essential for the study and the extension
of the already existing keloid is limited we think this is acceptable.