BackgroundMucopolysaccharidosis type III (MPS III), or Sanfilippo syndrome, is caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of heparan sulphate. Four MPS III types have been recognized, characterized by a large phenotypic heterogeneity. This is the first Spanish study describing the natural history of Sanfilippo patients (MPSIIIA, MPSIIIB and MPSIIIC), representing an essential step for understanding patient prognosis and for the establishment and application of future therapies.MethodsThis retrospective study aimed to establish the natural history of MPS III in Spain based on an extensive chronological data survey involving physicians and parents of 55 Spanish MPSIII patients. In addition to clinical description we report biochemical and molecular analysis already performed in the majority of cases.ResultsThe most frequent subtype was MPS IIIA (62%). Symptoms before diagnosis were speech delay in 85%, followed by coarse facial features in 78%, and hyperactivity in 65% of cases at a mean age of 3 years old. The median age at clinical and biochemical diagnosis for each MPS III subtype were as follows: IIIA 4.4 years (1.2 – 16 years), IIIB 3.1 years (1–29 years), and IIIC 6.3 years (3.4-22 years).45% of patients developed epilepsy at a median age of 8.7 (2.5 – 37) years old.Age of death for MPS IIIA patients was 15 years (11.5 – 26 years).Molecular analysis of our cohort reveals, as alluded to above, a great allelic heterogeneity in the three subtypes without clear genotype-phenotype correlations in most cases.ConclusionMPS IIIA is the most frequent subtype in Spanish Sanfilippo patients. Diagnosing physicians should consider Sanfilippo syndrome in children with non-specific speech delay, behavioural abnormalities, and/or mild dysmorphic features. We stress the importance of establishing early diagnosis procedures as soon as possible so as to be able to determine future short-term enzymatic or gene therapy treatments that can change the prognosis of the disease.

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B(12) metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B(12). The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients.

Niemann-Pick Types A and B (NPA/B) diseases are autosomal recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase (ASM) because of the mutations in the SMPD1 gene. Here, we provide a comprehensive updated review of already reported and newly identified SMPD1 variants. Among them, 185 have been found in NPA/B patients. Disease-causing variants are equally distributed along the SMPD1 gene; most of them are missense (65.4%) or frameshift (19%) mutations. The most frequently reported mutation worldwide is the p.R610del, clearly associated with an attenuated NP disease type B phenotype. The available information about the impact of 52 SMPD1 variants on ASM mRNA and/or enzymatic activity has been collected and whenever possible, phenotype/genotype correlations were established. In addition, we created a locus-specific database easily accessible at http://www.inpdr.org/genes that catalogs the 417 SMPD1 variants reported to date and provides data on their in silico predicted effects on ASM protein function or mRNA splicing. The information reviewed in this article, providing new insights into the genotype/phenotype correlation, is extremely valuable to facilitate diagnosis and genetic counseling of families affected by NPA/B.

Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.

BackgroundPhosphomannomutase deficiency (PMM2-CDG) is the most frequent congenital disorder of glycosylation. The cerebellum is nearly always affected in PMM2-CDG patients, a cerebellar atrophy progression is observed, and cerebellar dysfunction is their main daily functional limitation. Different therapeutic agents are under development, and clinical evaluation of drug candidates will require a standardized score of cerebellar dysfunction. We aim to assess the validity of the International Cooperative Ataxia Rating Scale (ICARS) in children and adolescents with genetically confirmed PMM2-CDG deficiency. We compare ICARS results with the Nijmegen Pediatric CDG Rating Scale (NPCRS), neuroimaging, intelligence quotient (IQ) and molecular data.MethodsOur observational study included 13 PMM2-CDG patients and 21 control subjects. Ethical permissions and informed consents were obtained. Three independent child neurologists rated PMM2-CDG patients and control subjects using the ICARS. A single clinician administered the NPCRS. All patients underwent brain MRI, and the relative diameter of the midsagittal vermis was measured. Psychometric evaluations were available in six patients. The Mann–Whitney U test was used to compare ICARS between patients and controls. To evaluate inter-observer agreement in patients’ ICARS ratings, intraclass correlation coefficients (ICC) were calculated. ICARS internal consistency was evaluated using Cronbach’s alpha. Spearman’s rank correlation coefficient test was used to correlate ICARS with NPCRS, midsagittal vermis relative diameter and IQ.ResultsICARS and ICARS subscores differed between patients and controls (p < 0.001). Interobserver agreement of ICARS was “almost perfect” (ICC = 0.99), with a “good” internal reliability (Cronbach’s alpha = 0.72). ICARS was significantly correlated with the total NPCRS score (rs 0.90, p < 0.001). However, there was no agreement regarding categories of severity. Regarding neuroimaging, inverse correlations between ICARS and midsagittal vermis relative diameter (rs −0.85, p = 0.003) and IQ (rs −0.94, p = 0.005) were found. Patients bearing p.E93A, p.C241S or p.R162W mutations presented a milder phenotype.ConclusionsICARS is a reliable instrument for assessment of PMM2-CDG patients, without significant inter-rater variability. Despite our limited sample size, the results show a good correlation between functional cerebellar assessment, IQ and neuroimagingFor the first a correlation between ICARS, neuroimaging and IQ in PMM2-CDG patients has been demonstrated.

Niemann-Pick disease (NPD) types A/B are both caused by a deficiency of lysosomal acid sphingomyelinase and display autosomal recessive inheritance. These two types of the disease were described according to the presence (type A) or absence (type B) of neurological symptoms. We present a molecular analysis of 19 Spanish NPD A/B patients and two from Maghreb. Eight of the patients had type A and 13 had type B NPD. All mutant SMPD1 alleles were identified, including 17 different mutations, 10 of which were novel. The only frequent mutations in the 21 NPD patients were c.1823_1825delGCC (p.R608del) (38%) and c.1445C>A (p.A482E) (9%). Genotype-phenotype correlations were established for most of the mutations and, in particular, the p.R608del-type B association was confirmed. This mutation accounts for 61.5% of the mutant alleles in the type B subgroup of patients. Expression studies performed on six of the identified mutations confirmed them to be disease-causing due to their low enzyme activity. An allele with a mutation affecting a non-canonical donor splice site produced only aberrant mRNAs corresponding to previously reported non-functional SMPD1 minor transcripts. This study is the first exhaustive mutational analysis of Spanish Niemann-Pick A/B disease patients.

Gaucher's disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-β-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-β-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-β-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.

BackgroundGaucher disease (GD) is due to deficiency of the glucocerebrosidase enzyme. It is panethnic, but its presentation reveals ethnicity-specific characteristics.MethodsWe evaluated the distribution, and clinical and genetic characteristics of GD patients in the Iberian Peninsula (IP). We analysed geographical distribution, demographic, genetic and clinical data, age at diagnosis, type, and years of therapy in 436 GD patients from the IP.ResultsThe prevalence of GD was 1/149,000 inhabitants; 88.3% were type 1, 6.7% type 2, and 5.0% type 3. The mean age at diagnosis in type 1 was 28.7 years. A total of 72.7% were classified as having mild forms, 25.5% moderate, and 1.7% severe. Anemia and thrombocytopenia were present in 56% and 55%, respectively. Bone disease and hepatomegaly were reported in 62% and 68%, respectively, and were more likely in asplenic than in non-splenectomized patients. Sixty-nine mutant alleles were identified, and five mutations accounted for 75% of the GBA alleles. Several patients described in our series had interesting phenotypes. A total of 58.7% of patients had received enzyme replacement therapy and 12.6% were treated with miglustat.ConclusionsA broad spectrum of GBA mutations is present in the IP, with 98.2% of type 1 GD being mild and 23.0% never treated. These data highlight genetic and phenotypic heterogeneities among geographic populations.