We describe vascular and endothelial function in addition to cerebral structure and function in two independent families with the same R373C mutation in the PROM1
gene. Carriers of the mutation were already reported to present clinical features of autosomal dominant macular dystrophy.7, 18
Human PROM1 is expressed by various stem and progenitor cells originating from diverse sources.23
Owing to the role of EPCs in vascular and endothelial function,24
we investigated these characteristics in five carriers of the R373C mutation. We found that the number of CECs, endothelial markers such as CD133, as well as the ability of EPCs to form endothelial colonies fell within the normal range in the tested patients. Endothelium-dependent and -independent functions, assessed by FMD, and the FMD:GTN ratio did not differ from controls. There was also no difference in IMT. The basic function and EPC regenerative resources would seem unaffected by the R373C mutation.
However, the number of circulating MPs was raised in patients compared with controls. MPs are membrane fragments shed by cells which have been activated by a variety of stimuli including serine proteases, inflammatory cytokines, growth factors and stress inducers.25
Clinical studies have revealed elevated plasma levels of MPs in multiple sclerosis, thrombotic thrombocytopenic purpura, coronary artery disease, hypertension, preeclampsia and diabetes.26, 27
Such data have led to the concept that endothelial MPs are key factors at the crossroads between inflammation, coagulation, proteolysis and vascular repair.28
Elevated circulating levels of MPs in our patients could be an indicator of either platelet, endothelial or leukocyte activation and could reflect a persistent state of endothelial dysfunction.
We looked also at the contribution of immature ECFCs to tubule growth. A significantly decreased ability of these cells to incorporate or adhere to the HUVECs was observed in carriers of the mutation. As this assay evaluates multiple cellular processes involved in blood vessel growth, EPC functional activity would appear impaired by the mutation. In turn, vascularization might be affected in the patients, at least under certain circumstances, perhaps those requiring neogenesis or repair.
Notably, the mutation carriers had microhaematuria and/or renal infections and a high number of miscarriages. Although there is no study reporting endothelial dysfunction in patients with haematuria and/or renal infections, several studies have reported that EPCs are numerically and functionally impaired in patients with acute29
or chronic renal failure,30
as compared with healthy subjects, and mechanisms such as inflammation or uraemic toxins have been given as potential explanations for the observed EPC dysfunction.30
More clear and direct is the relationship between miscarriages and possible endothelial dysfunction, given that development, maturation and maintenance of a neovascular network are necessary for successful haemochorial placentation as well as normal embryonic development and growth.31
Impairment in EPC functionality could thus affect placental angiogenesis and contribute to history of recurrent miscarriages in our patients. This requires further study in a larger group of patients, and might merit study in women with unexplained recurrent miscarriages.
CD133 has been also reported to mark several cell types with a variety of putative roles, including involvement in hippocampal neurogenesis16, 17
and diverse haematopoietic and extrahaematopoietic progenitive and regenerative functions.32, 33, 34
Carriers of the PROM1
mutation showed hippocampal and cerebral volumes within normal ranges for all patients from both kindreds. Three patients had small lesions in WM. Interestingly, the presence of MPs has also been reported to be positively associated with contrast-enhancing lesions on brain MRI in subjects with multiple sclerosis35
and furthermore, endothelial dysfunction is thought to have an important role in the pathogenesis of cerebral small-vessel disease especially in those patients with concomitant silent lacunar infarcts and ischaemic WM lesions.36
In line with this possible accelerated aging effect, the older member from the kindred B and one carrier of the PROM1 mutation in family A showed some memory disturbance and impairment in measures of executive functions in addition to small WM lesions. The remaining carriers in both families had average IQs and unimpaired performance on the other cognitive measures.
The three (of four) members of family A showed an empty sella turcica. The familial empty sella together with an eye phenotype could suggest a developmental defect of mesenchymal origin caused by mutation in the same gene.37
Familial empty sella has not, to our knowledge, been reported previously. Three patients had impaired olfaction.
All together, the elevated plasma levels of MPs, decreased ability of EPCs to incorporate into or adhere to tubules, the presence of empty sella turcica as well as leucoaraiosis and cognitive decline in the oldest mutation carriers suggest endothelial dysfunction that could be more pronounced or more evident with age. The R373C mutation results in a stable mutant protein. However, the mutant protein is not only mislocalized in retina, but it also interferes with the action of the normal protein,7
thereby accounting for its dominant mode of inheritance. Though we found no difference in the number of circulating EPCs or numbers of cells expressing CD133, the mutation may impair the function. For example, the mutation could render EPCs less effective in the process of angiogenesis or repair of damaged endothelium. Carriers of the R373C mutation showed no obvious greater risk of vascular dysfunction, but structural and functional microcirculation alterations tend to occur during ageing. Functional vascular examination was only undertaken in one of the two older patients as the second declined to take part in this test. Moreover, the presence of high blood pressure and high cholesterol, as well as leucoaraiosis,38
could be a sign of vascular alteration. Longitudinal study of more individuals with PROM1
mutation might be informative.
To our knowledge, this is the first study reporting a family with eye phenotype together with empty sella turcica, presence of microhematuria and possible endothelial dysfunction. In the case of the eye phenotype (macular dystrophy), the penetrance was complete although showing differences in severity. Penetrance in the other phenotypes was incomplete. Notable is the fact that none of the members in family B presented extra-ocular features, nor MRI findings (other than a few WM lesions), besides the cognitive decline in the older member, raising the possibility of organ-specific penetrance differences, perhaps related to organ-specific splicing differences of the PROM1
transcription regulation is rather complicated and poorly understood: it exhibits extensive splice variation39
with tissue-specific distribution,40
and in addition, transcripts can be regulated by epigenetic factors, as suggested by experiments with artificial in vitro
However, we cannot exclude the possibility that mutation in a second gene could be influencing the extra-ocular phenotype in family A, especially the presence of empty sella turcica.
The role of PROM1
in brain is still unclear. The gene, by itself or in combination with other genes, seems to have a role in the development of the brain, and/or cerebral recovery and repair.17, 41
The heterozygous mutation studied here results in a stable protein7
and is perhaps insufficient to completely inhibit its function in the brain. In contrast, recessive mutations arise from either frameshift or nonsense mutations5, 6
that result in the generation of a premature stop codon and truncated protein.5, 6, 42
In these cases, the homozygous patients display a severe eye phenotype, and polydactyly was also reported in one of the carriers, an additional symptom that confirms the existence of variable penetrance associated with PROM1
. No other documentation about vascular, endothelial or brain structures was reported.
The nature and size of our study does not permit definitive conclusions but leads to the suggestion that endothelial function may be affected in patients with the PROM1 R373C mutation, despite the apparently normal levels of EPCs. Further studies are needed to confirm these interesting findings that broaden the phenotype of PROM1 mutation, and inform our understanding of CD133 function.