Combined Molecular Microscopy for Therapy and Personalized Medication in Rare Anaemias Treatments

Anaemia, which is defined as a haemoglobin concentration less than 6,82 * 8,06
mmol/L depending on gender and age, affects 1.6 billion individuals worldwide.
Approximately 10% of these individuals are affected by rare anaemia (RA). This
disease group includes approximately 90 different types of red blood cell (RBC)
diseases, of which 80% are hereditary or congenital in nature. As the
pathophysiology of the majority of these RA is poorly understood, the
appropriate treatment is often ineffective or even lacking. Although the total
number of affected individuals is substantial, the diversity in the underlying
causes has resulted in limited interest from the pharmaceutical industry.

Recent studies indicate that several RA are associated with altered cellular
ion homeostasis. These deficiencies may directly cause the disease, as in
hereditary xerocytosis, overhydrated hereditary stomatocytosis, familial
pseudohyperkalaemia, cryohydrocytosis and certain types of spherocytosis.
There are scenarios in which RA may be due to structural RBC abnormalities due
to congenital membrane protein defects, e.g., hereditary spherocytosis,
haemoglobinopathies (e.g., sickle cell anaemia, thalassemia and glucose
transporter GLUT1 mutations, which cause paroxysmal exertion-induced
dyskinesias, inducing haemolytic anaemia by a cation leak.

A third category of RA associated with altered cellular ion homeostasis are
enzyme deficiencies (e.g., phosphofructokinase deficiency). Moreover, it
appears reasonable to assume that novel, unidentified ion homeostasis
disturbances may significantly contribute to anaemia pathophysiology and may
constitute an important group of undiagnosed cases of RA. The CoMMiTMent
Consortium has various objectives for the specific types of hereditary
haemolytic anaemia. (A) For the well-known and well-described types of
hereditary haemolytic anemia, such as sickle cell anaemia, treatment concepts
will be developed. (B) For other, more rare types of HHA, the pathophysiology
and pathways leading to ion channel disturbances will be investigated. (C) Last
but not least, for the yet undiscovered types of HHA, new diagnostic tools will
be developed. The UMC Utrecht will mainly focus on objectives B and C.

Together with the consortium partners in the CoMMiTMenT project we will
implement existing tools and/or laboratory tests, such as intracellular
calcium, sodium and potassium measurements as well as tools to measure ion
channel activities. We also will investigate the role of nanoparticles or
microvesicles, which are bud off from the plasma membrane of the red cell
during cellular stress, and might act as biomarkers for these specific
diseases. One of the triggers for the release of microvesicles from the red
cell membrane is an elevation of intracellular calcium and therefore these
microvesicles might act as biomarkers of ion channel disturbances of the red
cell. Microvesicles might also be involved in the comorbidities which patients
with hereditary haemolytic anaemia are suffering from, since microvesicles have
prothrombogenic and proimflammatoiry properties
.

The CoMMiTMenT consortium includes small and medium sized enterprises. Together
with the clinical partners, the consortium will develop a new diagnostic tool.
This tool will be called µCOSMOS and will be a combination of two already
existing techniques: Optofluidic Microscopy-based Cell Sorting (OMiCS) and
Scanning Ion Conductance Microscope (SICM). OMiCS optically measure cells in a
microfluidic system. Cells will be sorted with a laser based on their size,
shape or abnormal morphology. SICM then scans the surface of the sorted cells
with an electric probe. This measurement with SICM will be non-invasive and
will result in a topographic image of the red cell at a nanometer resolution.
Ultimately, the development of this µCOSMOS device will result in a system
which is able to select and study diseased red cells from a very heterogeneous
population of cells. Current diagnostic tools only study the total population
of red blood cells.

(i) identification and cellular characteristics of red blood cells from
patients with altered ion homeostasis and delivery of novel and easy-to-use
diagnostics for rare anemia*s, for patients with:
a) a known primary defect in the ion channels of the erythrocyte, e.g.
stomatocytosis
b) Secondary defect(s) of ion channels of the erythrocyte. For example,
unfunctional ion channels due to shortage of energy caused by metabolic
disorders (e.g. phosphofructokinase-defiency) or upregulated NMDA-receptors in
sickle cell anaemia causing influx of calcium25.
c) Patients with an unknown cause for haemolytic anaemia, of which
yet-unidentified ion channel disturbances may be the primary or secondary cause
of hemolysis.

(ii) identification of microvesicles as biomarkers or diagnostic tools and to
study their clinical relevance in (rare) haemolytic anaemias

The proposed study is a mono-center descriptive cohort study.

In this study patients with hereditary xerocytosis, overhydrated hereditary
stomatocytosis, familial pseudohyperkalaemia, cryohydrocytosis, certain types
of spherocytosis, hyperphosphatidylcholine haemolytic anaemia, sickle cell
anaemia, thalassemia syndromes, RBC enzymopathies (i.e., disorders of
glycolysis and glutathione metabolism) and patients with an unknown cause of
hereditary hemolytic anemia will be recruited from Academic partner hospitals
in Europe. The recruitment of patients will also take place in academic
hospitals in Europe we have international and academic relation with, inter
alia:

* University Medical Centre Utrecht, The Netherlands
* Consorci Institut d*Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS),
Barcelona, Spain
* Foundation IRCCS Ca* Granda Ospedale Maggiore Policlinico, Oncohematology
Unit, Physiopathology of Anemias Unit, Milan, Italy
* Centro Hospitalar e Universitario de Coimbra, Coimbra, Portugal

Shipping and transfer of patient material from hospitals across to the
University Medical Centre Utrecht, The Netherlands and vice versa will be
carried out according to the *Material Transfer Agreement' as shown in the K6.
CoMMiTMenT Consortium Agreement and K6. CoMMiTMenT Grant Agreeement* as shown
in the appendix. Patients in other national and international academic
hospitals will be recruited by their local physician.

Burden is limited to the donation of 41 ml blood, once of twice. If possibly,
all donations will be obtained during regular visits in the University Medical
Centre Utrecht. The current study will not be possible without the cooperation
of this specific group of patients. The patient does not benefit directly from
this study. However, we expect that this study leads to a better understanding
of the underlying pathophysiology of the rare anaemia he or she is suffering
from. On the long term this may lead to alternative treatments.