The diagnosis of HHT prompted a mutation analysis, which demonstrated a c. 1478 del G (p. C493SfsX25) in the exon 11 of the ENG
gene (see Olivieri et al6
for methods) (). The mutation identified a deletion of a single base, which introduces a stop codon. This kind of mutation, originating a truncated protein, is commonly assumed to be causative of the disease.
The sequence of polymerase chain reaction (PCR) product shows a deletion of 1 bp (c.1478delG (p. C493SfsX25)) located in exon 11 of ENG gene.
Then we studied the immunohistochemical expression of endoglin, TGF-β and Smad4 on the merkeloma cells (); the three proteins belong to the same pathway, relevant for blood vessel formation and maturation. Vessels were identified by studying CD31 and CD34. No evident vascular alterations were observed in the merkeloma (images not included).
Immunohistochemical staining of sections of merkeloma in the index case: (a) haematoxylin and eosin; (b) endoglin; (c) TGF-β; (d) Smad4.
To evaluate Smad4, TGF-β, CD105, CD31 and CD34 we performed an immunohistochemical analysis. The immunohistochemical method involved sequential application of primary antibody (Santa Cruz Biotechnology Inc, Santa Cruz, USA) to Smad4 (diluted 1:50), TGF-β (diluted 1:200), CD105 (diluted 1:50), CD31 (Novocastra Laboratories, Newcastle, UK, diluted 1:50) and CD34 (Neomarkers, Bioptica, Milan, Italy, diluted 1:30). We used the NovoLink Polymer Detection System (Novocastra Laboratories, Newcastle, UK).
Immunostaining was considered positive for all the antibodies analysed when at least 10% of neoplastic cells (for Smad4, TGF-β, CD105) and of endothelial cells (for CD31, CD34) were stained.
Tumour cells were positive for Smad4, weakly positive for TGF-β, and negative for CD105. Vasal endothelial cells were highly positive for CD105, CD31 and CD34. We did not observe remarkable immunohistochemical differences either between cancer and normal cells for CD105 and Smad4 of the same patient, or between the patient’s merkeloma and two merkeloma controls.