Definiendo la etiopatogénesis del síndrome de deficiencia de creatina cerebral

Resum

Creatine (Cr) plays an essential role for the maintenance of ATP levels in tissues with high energy demand, such as brain. Defects affecting the Cr biosynthesis (arginine: glycine amidino transferase (AGAT) and guanidinoacetate methyltransferase (GAMT) or the transporter (CRT1) are known as creatine deficiency syndrome (CDS). In all these disorders the common hallmark is cerebral creatine deficiency which causes a wide range of neurological manifestations. They constitute a group of rare inherited metabolic diseases which pathophysiology is still under investigation. The aims of this work have been improving the diagnosis of these defects studying the patients at biochemical and genetic level, and exploring the impact of our cohort of natural CDS mutants on different variables of cellular metabolic stress. Nine 9 CDS patients have been identified in a prospective screening based on abnormal metabolite excretion or decreased creatine signal in brain 1H-MRS in a cohort of patients with mental retardation (MR) and/or suspected metabolic disorder. Six CRT1 and three GAMT cases were identified. Confirmatory diagnosis was assessed by measuring creatine uptake and GAMT activity in cultured fibroblasts, respectively. Direct sequencing analysis of SLC6A8 gene revealed six sequence variations, four of them novel: c.462G>A (p.W154X); c.1079_1081delTCT (p.F360del); c.1210G>C (p.A404P); c.1396G>A (p.G466R). The mutational spectrum of the GAMT gene included 5 allelic variants, 3 of them novel: c.145delT (p.Y49fs) which appeared in homozygous fashion and c.316C>T (p.Q106X) and c.407C>T (p.T136M), both in heterozygous fashion. All changes identified in both genes were characterised in silico, in vitro and in vivo, and all of them fulfilled several criteria to be considered as pathogenic mutations. Our results confirm the presence of CDS patients in a broad population of neurological patients including those with MR of unknown aetiology highlighting the importance of screening for these potentially treatable disorders. Furthermore, we analyzed the impact of these CDS mutants on parameters of metabolic cellular stress, such as ROS production, aberrant cell proliferation, activation of stress mediated signalling pathways and apoptosis. An increase of intracellular ROS content and percentage of apoptotic cells as well as an altered profile in cell cycle progression and activation of p53 and p38MAPK were detected in some CRT1 and GAMT fibroblasts, mostly those carrying null alleles. Based on intracellular creatine levels, contribution of energy depletion to metabolic stress could only be demonstrated in selected CRT1 cells. So although genotype-phenotype correlation is not straightforward, this association seems to be clearer in CRT1 patients. We consider that these studies exert an essential impact in clinics because they support the potential of reducing intracellular ROS and oxidative-stressinduced apoptosis as a novel strategy for the treatment of these disabling diseases.