Pedro Ruiz-Sala

A novel congenital disorder of glycosylation type without central nervous system involvement caused by mutations in the phosphoglucomutase 1 gene

Recent years have seen great advances in our knowledge of congenital disorders of glycosylation (CDG), a clinically and biochemically heterogeneous group of genetic diseases caused by defects in the synthesis (CDG-I) or processing (CDG-II) of glycans that form glycoconjugates. This paper reports a new subtype of non-neurological CDG involving the impaired cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. A patient presented with muscle fatigue, elevated creatine kinase, growth hormone deficiency, and first branchial arch syndrome. These findings, together with the abnormal type II plasma transferrin isoform profile detected, was compatible with a CDG. Functional testing and clinical analyses suggested a deficiency in the interconversion of glucose-1-phosphate and glucose-6-phosphate catalyzed by phosphoglucomutase (PGM1), a defect previously described as glycogenosis type XIV (GSDXIV, MIM 612934). PGM1 activity in patient-derived fibroblasts was significantly reduced, as was the quantity of immunoreactive PGM1 protein (Western blot assays). Mutation analysis of PGM1 and subsequent functional analysis investigating transient expression of PGM1 in immortalized patient fibroblasts, followed by ex vivo splicing assays using minigenes, allowed the characterization of two novel pathogenic mutations: c.871G>A (p.Gly291Arg) and c.1144 + 3A>T. The latter represents a severe splicing mutation leading to the out-of-frame skipping of exon 7 and the formation of a truncated protein (p.Arg343fs). MALDI mass spectra of permethylated protein N-glycans from the patient’s serum suggested a marked hypoglycosylation defect. The present findings confirm that, in addition to a rare muscular glycolytic defect, PGM1 deficiency causes a non-neurological disorder of glycosylation.

Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option

Pyridoxine‐dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α‐aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease.

Marked mitochondrial DNA depletion associated with a novel SUCLG1 gene mutation resulting in lethal neonatal acidosis, multi-organ failure, and interrupted aortic arch

The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations.

Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations.

A new case of creatine transporter deficiency associated with mild clinical phenotype and a novel mutation in the SLC6A8 gene

Patricia Alcaide, Pilar Rodriguez-Pombo, Pedro Ruiz-Sala, Isaac Ferrer, Pedro Castro, Yolanda Ruiz Martin, BegoÇa Merinero, Magdalena Ugarte 1 Centro de Diagn stico de Enfermedades Moleculares, Departamento Biolog a Molecular CBM-SO, CSIC-UAM, Universidad Aut noma de Madrid, Madrid, Spain. 2 Centro de Investigaci n Biom dica en Red de Enfermedades Raras (CIBERER), Madrid, Spain. 3 Servicio de Neuropediatr a, Hospital Gregorio MaraÇ n, Madrid, Spain. Correspondence to: mugarte@cbm.uam.es

Thirteen Years Experience with Selective Screening for Disorders in Purine and Pyrimidine Metabolism

Purine and pyrimidine disorders represent a heterogeneous group with variable clinical symptoms and low prevalence rate. In the last thirteen years, we have studied urine/plasma specimens from about 1600 patients and we have identified 35 patients: eight patients with adenylosuccinate lyase deficiency, eight patients with hypoxanthine-guanine phosphoribosyltransferase deficiency, one patient with purine nucleoside phosphorylase deficiency, ten patients with xanthine dehydrogenase deficiency, six patients with molybdenum cofactor deficiency and two patients with dihydropyrimidine dehydrogenase deficiency. Despite low incidence of these diseases, our findings highlight the importance of including the purine and pyrimidine analysis in the selective screening for inborn errors of metabolism in specialized laboratories, where amino acid and organic acid disorders are simultaneously investigated.

Determination of urinary alpha-aminoadipic semialdehyde by LC–MS/MS in patients with congenital metabolic diseases

This paper describes a full detailed high performance liquid chromatography/tandem mass spectrometry method for the identification and quantification of human urine alpha-aminoadipic semialdehyde, biomarker of pyridoxine-dependent epilepsy. The ionization mode of the electrospray interface was negative and the metabolite was detected in the multiple reaction monitoring mode. Intra-day and inter-day laboratory precision were 4.64% and 7.30%, respectively, total run time was 3.5min. The calibration curve was linear between 0.25 and 10nmol with a correlation coefficient of the calibration line (R(2)≥0.9984); the limit of quantification was 0.25nmol within the control group. This simple, fast, high reproducible and robust procedure facilitates a rapid diagnosis of patients with pyridoxine-dependent epilepsy and can also be used to confirm the elevated urinary alpha-aminoadipic semialdehyde excretion in patients with other metabolic diseases as molybdenum cofactor and isolated sulphite oxidase deficiencies.

Urine oligosaccharide tests for the diagnosis of oligosaccharidoses

Abstract This review discusses the development of capillary electrophoresis with laser-induced detection and mass spectrometry techniques for the analysis of urinary oligosaccharides to screen for human oligosaccharidoses and related disorders. Capillary electrophoresis is suitable for the analysis of urinary oligosaccharides. It has a high resolution efficiency, and when it is coupled to a laser-induced fluorescence detector system, it offers an optimal analytical sensitivity. Mass spectrometry techniques have evolved as powerful tools for glycan analyses, are important tools for the analysis of oligosaccharide structures, and offer precise results, analytical versatility, very high sensitivity, high precision and high speed. Mass spectrometry is tolerant of mixtures and is a natural choice for the analysis of this class of molecules. The urine oligosaccharide profiles from healthy controls and patients diagnosed with different lysosomal storage diseases (fucosidosis, α-mannosidosis, GM1 gangliosidosis, GM2 gangliosidosis type Sandhoff, glycogen storage disease type 2 and 3, aspartylglucosaminuria, Schindler disease and galactosialydosis) and related disorders (glycogen storage diseases) are presented. In summary, these procedures are powerful tools for the rapid identification and characterization of these disorders. In all cases, the oligosaccharide profiles are strongly informative, identified the abnormal compounds, and are good alternatives to traditional screening tests that use thin layer chromatography. These tests have great sensibility and resolution and are automatable, enabling extended screening of these diseases, both as a first step in diagnosis or for confirming the pathogenicity of mutations that may be detected with next generation sequencing techniques.