Beckwith-Wiedemann Syndrome: Potassium Ascorbate with Ribose Therapy in a Syndrome with High Neoplastic Risk

Patients and Methods

Patient 1. A female, born from twins delivered at the 35th week of pregnancy, presented overgrowth at birth, diastasis recti, facial nevus flammeus, macroglossia, epicanthus, ear lobe pits, bi-temporal diameter reduce, tendency for neonatal hypoglycemia, interatrial defect, and mild asymmetry of the face, chest and gluteal muscles and hepatomegaly. These is a familiar case of BWS. The mutation involved results in the hypermethylation of Imprinting Centre 1 (IC1) region, loss of insulin growth factor (IGF2/H19) imprinting and fully penetrant BWS phenotype which is maternally transmitted (7). This mutation is also associated with high neoplastic risk, specifically for Wilm's tumor, of 25% (8). Wilm's tumor at stage I was discovered by ultrasound when the patient was 11 months old. She had surgery and follow-up examinations at our clinic every 3 months. We evaluated the OS biomarker profile for the first time when she was 4 years and 5 months old, finding significantly higher levels than controls (twenty healthy children from 1 month to 5 years). PAR therapy (300 mg of Potassium Bicarbonate and 150 mg of Ascorbic Acid in aqueous solution every day) started immediately after.

Patient 2. A female, born at the 36th week of pregnancy, presented neonatal overgrowth, macroglossia, macrostomia, asymmetry in the root of the thighs, crumpled helix in the right ear, small ear lobe pits, tendency for neonatal hypoglycemia, interventricular defect, patent foramen ovale, persistent Botallo duct, voluminous umbilical cord, polyhydramnios at 6 months of pregnancy which later regressed. The patient had a normal karyotype, but molecular diagnosis showed an impairment of Imprinting Centre 2 (IC2), with an hypomethylation of KvDMR1 that is linked to a low neoplastic risk (8, 9). We evaluated an OS biomarker profile at a neonatal age (twenty children from 1 month to 5 years were used as controls) and once OS was ascertained, PAR therapy immediately started (300 mg of Potassium Bicarbonate and 150 mg of Ascorbic Acid in aqueous solution every day).

Patient 3. The patient was 38 years old and the mother of patient 1. Anamnesis revealed neonatal overgrowth and macroglossia. At birth, a left side congenital torticollis was diagnosed, later defined as neck hyperplasia. Molecular diagnosis showed the same mutation as that of her daughter. The mutation resulted in the hypermethylation of the IC1 region with loss of IGF2/H19 imprinting (7). High levels of OS biomarkers were ascertained at the age of 32 years. Twenty adults (aged from 25 to 40 years) were used as controls. PAR therapy (300 mg of Potassium Bicarbonate and 150 mg of Ascorbic Acid in aqueous solution twice a day) immediately started and a decrease in OS was observed. This mutation has a high neoplastic risk (>20%) (8), although the patient has not yet developed any neoplasms.

Methods. Heparinized blood samples were drawn and a reliable OS biomarker profile was elaborated at the time of diagnosis. Blood samples were immediately centrifuged (1000 × g for 10 min). The plasma was stored in plastic metal-free containers at −80°C until analyses, which were performed at the Laboratory of Oxidative Stress of the University of Siena. Forty healthy individuals of the same age as our patients, i.e. 20 adults (aged from 25 to 40 years) and 20 children (aged from 1 month to 5 years), were used as controls (Table II). The biomarker profile included non-protein-binding iron (NPBI), total hydroperoxides (TH) advanced oxidation protein products (AOPP), isoprostanes (IP₂), and carbonyl groups (COG). NPBI, being redox cycling active, has pro-oxidant properties because it can enter into the Fenton reaction producing a hydroxyl radical, the most oxidizing molecule in biological systems (10). TH are indices of overall FR attack, because they are indicative of intermediate oxidative products of lipids, peptides, and amino acids. Lipid and protein hydroperoxide, in the presence of traces of free iron, produce several secondary reactive radical species which can be measured collectively as organic hydroperoxide (11). AOPP are terminal products of protein exposure to FR. IP₂ are recognized as the most reliable lipoperoxidation products. IP₂ are generated by FR attack on to polyunsaturated fatty acids. Their accumulation in human tissues is a cause of cellular dysfunction. Recently there has been striking evidence implicating IP₂ in cancer development (12, 13).

NPBI was measured with the method described by Paffetti (14). This method is based on preferential chelation of NPBI by a large excess of the low-affinity ligand nitrilotriacetic acid (14). TH production was measured with a d-ROMs Kit (Diacron, Rome, Italy) as described by Buonocore (11). This method makes it possible to estimate TH present in a 10 μl blood sample using a spectrophotometric procedure. Hydroperoxidic groups are attacked by iron, decompartmentalized from the transport protein in 1 ml acetate buffer at pH 4.8, to catalyze formation of reactive oxygen metabolite by Fenton's reaction. The peroxy and alkoxy radicals produced, the quantities of which are directly proportional to peroxides present in the plasma, are trapped chemically by 10 μl chromogen in an electron transfer process leading to the formation of the radical cation of this chromogen. The purple color resulting from this reaction over time was then monitored in a UV-VIS spectrophotometer (λ 16; Perkin-Elmer, Norwalk, CT, USA) at 505 nm. The results were expressed in conventional units, called Carr units; 1 Carr unit is equal to a concentration of 0.08 mg/l hydrogen peroxide. TH represents a measure of overall oxidative stress, given that they are the intermediate oxidative products of lipids, peptides, and amino acids(11). IP₂ was determined using gas chromatography with mass spectrometry (GC-MS) according to the methodology of Milne and Morrow (15).

Furthermore, we measured thiols, usually considered markers of antioxidant power, in the adult patient because they are normally lower in OS-related disease. Thiols were measured with an SHp Test (Diacron) using the spectrophotometric procedure of Ellman (16). We used the method of Witko-Sarsas for AOPP, and Protein Carbonyl Assay Kit (Cayman Chemical, MI, USA) for COG.

We observed our patients quarterly over a range of times according to the different symptoms they showed and we submitted them to clinical and instrumental examinations as suggested in the literature (1). Finally, every three months OS biomarker profile of blood samples were carried out after a daily administration of PAR at the dosage of 300 mg of Potassium Bicarbonate and 150 mg of Ascorbic Acid in aqueous solution. The present study was approved by the local Ethics Committee of University Hospital of Siena.