Investigating phosphatidylserine metabolism in Lenz-Majewski syndrome

We recently uncovered the genetic cause of a syndrome (Lenz-Majewski syndrome or LMS), which is characterized by progressive problems in the skeleton such as excessive bone density and bone malformations including, shortness and fusing of fingers and toes, together with tooth, skin and brain defects. The condition is caused by an anomaly in the production of phospholipids, which are an essential component of all human cell membranes. The study identified mutations in an enzyme that produces one specific phospholipid (phosphatidylserine, which makes up 10% or less of the total), . The mutations activate the enzyme, causing it to manufacture too much phosphatidylserine, which leads on to cause the bone problems described above. The exact mechanism is not yet understood and this proposal seeks to clarify the key events at a cellular and biochemical level. To do this, we propose to use several different model systems. These include cell lines derived from human patients, mice and zebrafish that have been engineered to have the relevant genes inactivated, or altered to mimic the most common human mutation. We will study these models first to investigate how their lipid composition has changed as a consequence of the mutation/gene disruption. We will look at how gain or loss-of-function variants affect bone formation, both during embryonic/fetal development and through to adult life. It will be very important to determine if loss of function has an opposite effect to gain of function, as this may provide new insights into the control of bone formation and turnover throughout life. We will also make a detailed investigation of the different cell types that are involved in regulating bone formation and maintenance. Our findings will be very useful for a better understanding of the role of phosphatidylserine in normal bone development but also to open up the potential for developing therapies. In this project, we use our model systems to test the effects of specifically targeted drugs, as well as using a high-throughput screening system designed to identify novel compounds that can restore bone metabolism through affecting lipid metabolism. Resulting drugs may have implications for treatment of bone defects from osteoporosis to osteoarthritis.

We aim to understand the biochemical interaction between disordered phosphatidylserine (PS) synthesis and bone metabolism as highlighted in Lenz-Majewski syndrome (LMS). We will employ UPLC tandem mass spectrometry to investigate lipid composition and the production of specific PS isoforms, first in patient fibroblasts and lymphoblasts cell lines and later in cells derived from animal models. Biochemical analysis will be performed on osteoclasts derived from LMS whole blood.
We will investigate PS metabolism in mice with reduced PS synthase enzyme activity and create knock in mice mimicking the common human mutation. This will use standard methods including homologous recombination in ES cells and chimera production. Zebrafish mutants will be created using the Tol2 kit. This method facilitates the rapid construction of mutants allowing insertion or replacement of different elements in a modular form e.g. promoters or protein tags. We will use different promoters to drive expression in specific cell lineages to dissect which are relevant for PS in disordered bone development.
Osteogenesis and later skeletal development will be investigated in mice and fish using standard in situ hybridization and histological techniques. Postnatal skeletal analysis on mice up to 1 year old will be monitored using DEXA and microCT, the latter also used to investigate the fish skeleton.
We will use the most appropriate model system to test the ability to modify PS biosynthesis using molecules or compounds that might have therapeutic benefit. It is likely that this will be a patient, fish or mouse cell line grown in 384-well microtitre plates in the presence of deuterated stable isotopes derivatives of fatty acids/serine. Cells can then be assayed following treatment with an array of test molecules, using UPLC tandem mass spectrometry. This final aim is specifically relevant clinically for treatment of LMS patients and potentially in more generalised bone disease.

Disorders of the skeleton range from severe birth defects/rare syndromes, to common late onset disorders of bone function, such as osteoporosis, rheumatoid-/osteoarthritis to osteopetrosis. These disorders represent an ever increasing burden in our ageing population. Osteoporosis, is a common defect in bone turnover tissue homeostasis, affecting over 20% of women and 4% of men, with an estimated $17bn/yr cost in the United States alone. However, there are more than 100 distinct rare genetic skeletal disorders that have been described, and >1 in 500 people are affected by one of these conditions. The key metabolic factors influencing bone development are still far from clearly understood and these rare conditions offer unique and precious insights into otherwise hidden mechanisms. Our work on Lenz-Majewski syndrome (LMS) will greatly clarify the important role played by phosphatidylserine (PS) and other phospholipids in health and disease. This is not only important for sufferers of bone anomalies but may also be important knowledge for the PS supplement market advertised to benefit cognition and exercise induced stress.

Specific Beneficiaries:

For the patients: Both current and future patients with LMS and those with related bone disorders, will gain from a fuller understanding of the mechanism and natural history of their devastating condition. As part of this work, we will investigate potential drugs/small molecules that can be used to modulate phoshatidylserine metabolism, with a view to identify an effective therapy.

For researchers: Our findings will expand the respective fields of both lipid and bone metabolism. We will provide new tools to research novel aspects of these fields, in particular creating the mouse knock-in of the human missense mutation, creating transgenic zebrafish expressing wild-type and mutant Ptdss1, with a range of cell lineage specific promoters. Through our use of mass spectroscopy, we are developing the field of lipidometry, which is likely to be an important and expanding tool in the study of metabolic disease. Therefore we are developing several new transferrable research tools and skills.

For clinicians: We have recently discovered a molecular diagnosis. This work will inform about the natural progression of the disease at a cellular and biochemical level. We will uncover potential treatments with a view to improving the patient's wellbeing and reducing the burden of care. It is also likely to be of great interest to orthopedic, dental, maxilo-facial and plastic surgeons, as well as those with an interested in prosthetics, for the purpose of bone repair, regeneration, replacement and tissue engineering fields. Here there is already promising research on phophatidylserine and phosphatidylserine-mimicking coating technology in osteointegrative biomaterials.