piRNA pathway evolution beyond gonad context: Perspectives from apicomplexa and trypanosomatids.

piRNAs function as genome defense mechanisms against transposable elements insertions within germ line cells. Recent studies have unraveled that piRNA pathways are not limited to germ cells as initially reckoned, but are instead also found in non-gonadal somatic contexts. Moreover, these pathways have also been reported in bacteria, mollusks and arthropods, associated with safeguard of genomes against transposable elements, regulation of gene expression and with direct consequences in axon regeneration and memory formation. In this Perspective we draw attention to early branching parasitic protozoa, whose genome preservation is an essential function as in late eukaryotes. However, little is known about the defense mechanisms of these genomes. We and others have described the presence of putative PIWI-related machinery members in protozoan parasites. We have described the presence of a PIWI-like protein in Trypanosoma cruzi, bound to small non-coding RNAs (sRNAs) as cargo of secreted extracellular vesicles relevant in intercellular communication and host infection. Herein, we put forward the presence of members related to Argonaute pathways in both Trypanosoma cruzi and Toxoplasma gondii. The presence of PIWI-like machinery in Trypansomatids and Apicomplexa, respectively, could be evidence of an ancestral piRNA machinery that evolved to become more sophisticated and complex in multicellular eukaryotes. We propose a model in which ancient PIWI proteins were expressed broadly and had functions independent of germline maintenance. A better understanding of current and ancestral PIWI/piRNAs will be relevant to better understand key mechanisms of genome integrity conservation during cell cycle progression and modulation of host defense mechanisms by protozoan parasites.

Clinical and molecular diagnosis of non-phosphomannomutase 2 N-linked congenital disorders of glycosylation in Spain.

The congenital disorders of glycosylation (CDG) are defects in glycoprotein and glycolipid glycan synthesis and attachment. They affect multiple organ/systems, but non-specific symptoms render the diagnosis of the different CDG very challenging. Phosphomannomutase 2 (PMM2)-CDG is the most common CDG, but advances in genetic analysis have shown others to occur more commonly than previously thought. The present work reports the clinical and mutational spectrum of 25 non-PMM2 CDG patients. The most common clinical symptoms were hypotonia (80%), motor or psychomotor disability (80%) and craniofacial dysmorphism (76%). Based on their serum transferrin isoform profile, 18 were classified as CDG-I and 7 as CDG-II. Pathogenic variations were found in 16 genes (ALG1, ALG6, ATP6V0A2, B4GALT1, CCDC115, COG7, DOLK, DPAGT1, DPM1, GFPT1, MPI, PGM1, RFT1, SLC35A2, SRD5A3, and SSR4). Overall, 27 variants were identified, 12 of which are novel. The results highlight the importance of combining genetic and biochemical analyses for the early diagnosis of this heterogeneous group of disorders.

Four Years' Experience in the Diagnosis of Very Long-Chain Acyl-CoA Dehydrogenase Deficiency in Infants Detected in Three Spanish Newborn Screening Centers.

Identification of very long-chain acyl-CoA dehydrogenase deficiency is possible in the expanded newborn screening (NBS) due to the increase in tetradecenoylcarnitine (C14:1) and in the C14:1/C2, C14:1/C16, C14:1/C12:1 ratios detected in dried blood spots. Nevertheless, different confirmatory tests must be performed to confirm the final diagnosis. We have revised the NBS results and the results of the confirmatory tests (plasma acylcarnitine profiles, molecular findings, and lymphocytes VLCAD activity) for 36 cases detected in three Spanish NBS centers during 4 years, correlating these with the clinical outcome and treatment. Our aim was to distinguish unambiguously true cases from disease carriers in order to obtain useful diagnostic information for clinicians that can be applied in the follow-up of neonates identified by NBS.Increases in C14:1 and of the different ratios, the presence of two pathogenic mutations, and deficient enzyme activity in lymphocytes (<12% of the intra-assay control) identified 12 true-positive cases. These cases were given nutritional therapy and all of them are asymptomatic, except one. Seventeen individuals were considered disease carriers based on the mild increase in plasma C14:1, in conjunction with the presence of only one mutation and/or intermediate residual activity (18-57%). In addition, seven cases were classified as false positives, with normal biochemical parameters and no mutations in the exonic region of ACADVL. All these carriers and the false positive cases remained asymptomatic. The combined evaluation of the acylcarnitine profiles, genetic results, and residual enzyme activities have proven useful to definitively classify individuals with suspected VLCAD deficiency into true-positive cases and carriers, and to decide which cases need treatment.

The homozygous R504C mutation in MTO1 gene is responsible for ONCE syndrome.

We report clinical and biochemical finding from three unrelated patients presenting ONCE (Optic Neuropathy, Cardiomyopathy and Encephalopathy with lactic acidosis and combined oxidative phosphorylation deficiency) syndrome. Whole-exome sequencing (WES) of the three patients and the healthy sister of one of them was used to identify the carry gene. Clinical and biochemical findings were used to filter variants, and molecular, in silico and genetic studies were performed to characterize the candidate variants. Mitochondrial DNA (mtDNA) defects involving mutations, deletions or depletion were discarded, whereas WES uncovered a double homozygous mutation in the MTO1 gene (NM_001123226:c.1510C>T, p.R504C, and c.1669G>A, p.V557M) in two of the patients and the homozygous mutation p.R504C in the other. Therefore, our data confirm p.R504C as pathogenic mutation responsible of ONCE syndrome, and p.V557M as a rare polymorphic variant.

Secondary coenzyme Q10 deficiencies in oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders.

We evaluated the coenzyme Q10 (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation.

Assessment of resting energy expenditure in pediatric mitochondrial diseases with indirect calorimetry.

BACKGROUND & AIMS: Mitochondrial diseases (MD) are the most frequent inborn errors of metabolism. In affected tissues, MD can alter cellular oxygen consumption rate leading to potential decreases in whole-body resting energy expenditure (REE), but data on pediatric children are absent. We determined, using indirect calorimetry (IC), whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ) and REE in pediatric patients with MD and healthy controls. Another goal was to assess the accuracy of available predictive equations for REE estimation in this patient population. METHODS: IC data were obtained under fasting and resting conditions in 20 MD patients and 27 age and gender-matched healthy peers. We determined the agreement between REE measured with IC and REE estimated with Schofield weight and FAO/WHO/UNU equations. RESULTS: Mean values of VO2, VCO2 (mL·min-1·kg-1) or RQ did not differ significantly between patients and controls (P = 0.085, P = 0.055 and P = 0.626 respectively). Accordingly, no significant differences (P = 0.086) were found for REE (kcal·day-1 kg-1) either. On the other hand, although we found no significant differences between IC-measured REE and Schofield or FAO/WHO/UNU-estimated REE, Bland-Altman analysis revealed wide limits of agreement and there were some important individual differences between IC and equation-derived REE. CONCLUSIONS: VO2, VCO2, RQ and REE are not significantly altered in pediatric patients with MD compared with healthy controls. The energy demands of pediatric patients with MD should be determined based on IC data in order to provide the best possible personalized nutritional management for these children.

Presentación atípica y diagnóstico tardío en un caso de déficit primario de ácidos biliares / Atypical presentation and delayed diagnosis in a case of primary bile acid synthesis disorder

No disponible

Estudio epidemiológico de las enfermedades metabólicas con homocistinuria en España / Epidemiological study of the metabolic diseases with homocystinuria in Spain

Objetivos: Conocer la prevalencia en España de los diferentes errores congénitos del metabolismo que presentan homocistinuria y establecer las medidas oportunas para garantizar su prevención, diagnóstico y tratamiento, en aquellos casos posibles. Material y métodos: En abril 2009 se realizó una encuesta nacional de carácter transversal mediante cuestionario enviado a 35 centros, en los que se atiende a pacientes infantiles y adultos. La finalidad de la encuesta era establecer la prevalencia en ese momento recogiendo el histórico de pacientes que cada centro tuviera documentados. Resultados: A través de los cuestionarios respondidos por 25 médicos de 16 centros, se han identificado 75 pacientes: 41 defectos de transulfuración (uno fallecido), 27 de remetilación (6 fallecidos) y 7 sin diagnóstico etiológico definitivo. La edad de diagnóstico muestra una amplia variación, en 18 casos había más de un hermano afectado. Las manifestaciones clínicas más graves inciden en el grupo de los pacientes afectados de trastornos de la remetilación. Destaca el alto porcentaje de déficit cognitivo, seguido de la patología de cristalino; casi la mitad de los pacientes presentan trastornos neurológicos, es elevada la afectación vascular en los adultos con deficiencia de CBS; las opciones terapéuticas más utilizadas han sido el ácido fólico, la hidroxicobalamina y la betaína. Conclusiones: A la vista de estos resultados, y en especial del escaso número de deficiencias de CBS detectadas, se concluye la necesidad de implantar el cribado neonatal para la homocistinuria clásica y asegurar la puesta en marcha del proceso diagnóstico oportuno en todos los pacientes de riesgo(AU)

Objectives: To determine the prevalence of homocystinuria in Spain and to establish the measures and mechanisms to ensure its prevention, diagnosis and treatment. Material and methods: A national cross-sectional survey was conducted by means of a questionnaire sent to 35 hospitals in which children and adult patients are treated. Results: Using the questionnaires submitted by 25 physicians from 16 centres, 75 patients were identified: 41 transsulphuration defects (one deceased), 27 remethylation (six deaths) and 7 without a syndromic diagnosis. The age at diagnosis varied widely, and 18 cases had more than one sibling affected. The more severe clinical manifestations involved the patients with remethylation defects. There was a high percentage of cognitive impairment, followed by lens diseases. Almost half of the patients had neurological disorders. There was increased vascular involvement in CBS-deficient adults. The therapeutic options most used were, folic acid, hydroxycobalamin and betaine. Conclusions: In view of these results and especially the small number of CBS deficiencies detected, we conclude that there is a need to introduce newborn screening for classical homocystinuria and ensure implementation of an appropriate diagnostic workup in all patients at risk(AU)

Mitochondrial tRNA valine as a recurrent target for mutations involved in mitochondrial cardiomyopathies.

The aim of this study was to identify the genetic defect in two patients having cardiac dysfunction accompanied by neurological symptoms, and in one case MRI evidence of cortical and cerebellar atrophy with hyperintensities in the basal ganglia. Muscle biopsies from each patient revealed single and combined mitochondrial respiratory chain deficiency. The complete mtDNA sequencing of both patients revealed two transitions in the mitochondrial tRNA(Val) gene (MT-TV) (m.1628C>T in Patient 1, and m.1644G>A in Patient 2). The functional and molecular analyses reported here suggest that the MT-TV gene should be routinely considered in the diagnosis of mitochondrial cardiomyopathies.

Clinical, molecular and biochemical characterization of nine Spanish families with Conradi-Hünermann-Happle syndrome: new insights into X-linked dominant chondrodysplasia punctata with a comprehensive review of the literature.

BACKGROUND: Conradi-Hünermann-Happle syndrome (CDPX2, OMIM 302960) is an inherited X-linked dominant variant of chondrodysplasia punctata which primarily affects the skin, bones and eyes. CDPX2 results from mutations in EBP (emopamil binding protein), and presents with increased levels of sterol precursors 8(9)-cholesterol and 8-dehydrocholesterol. OBJECTIVES: To expand the understanding of CDPX2, clinically, biochemically and genetically. METHODS: We present one of the largest series reported to date, including 13 female patients belonging to nine Spanish families. Patients were studied biochemically using gas chromatography-mass spectrometry, genetically using polymerase chain reaction and in their methylation status using the HUMARA assay. RESULTS: In our cases, there was a clear relationship between abnormal sterol profile and the EBP gene mutation. We describe three novel mutations in the EBP gene. EBP mutations were inherited in three out of nine families and were sporadic in the remaining cases. CONCLUSIONS: No clear genotype-phenotype correlation was found. Patients' biochemical profiles did not reveal a relationship between sterol profiles and severity of disease. A skewed X-chromosome inactivation may explain the clinical phenotype in CDPX2 in some familial cases.

[Epidemiological study of the metabolic diseases with homocystinuria in Spain]. / Estudio epidemiológico de las enfermedades metabólicas con homocistinuria en España.

OBJECTIVES: To determine the prevalence of homocystinuria in Spain and to establish the measures and mechanisms to ensure its prevention, diagnosis and treatment. MATERIAL AND METHODS: A national cross-sectional survey was conducted by means of a questionnaire sent to 35 hospitals in which children and adult patients are treated. RESULTS: Using the questionnaires submitted by 25 physicians from 16 centres, 75 patients were identified: 41 transsulphuration defects (one deceased), 27 remethylation (six deaths) and 7 without a syndromic diagnosis. The age at diagnosis varied widely, and 18 cases had more than one sibling affected. The more severe clinical manifestations involved the patients with remethylation defects. There was a high percentage of cognitive impairment, followed by lens diseases. Almost half of the patients had neurological disorders. There was increased vascular involvement in CBS-deficient adults. The therapeutic options most used were, folic acid, hydroxycobalamin and betaine. CONCLUSIONS: In view of these results and especially the small number of CBS deficiencies detected, we conclude that there is a need to introduce newborn screening for classical homocystinuria and ensure implementation of an appropriate diagnostic workup in all patients at risk.

Enfermedad de Niemann-Pick tipo C: desde una colestasis neonatal hacia un deterioro neurológico. Variabilidad fenotípica / Niemann-Pick type C disease: From neonatal cholestasis to neurological degeneration. Different phenotypes

Introducción: La enfermedad de Niemann-Pick tipo C está causada por un defecto en el transporte intracelular de colesterol que produce un acúmulo de lípidos en los lisosomas de diferentes tejidos. Es una enfermedad rara, debida generalmente a mutaciones en el gen NPC1 y solo unos pocos casos se asocian a mutaciones en el gen NPC2. Frecuentemente se manifiesta en la edad pediátrica, presentando gran variabilidad en las manifestaciones clínicas. La enfermedad conduce a un deterioro neurológico con diferentes síntomas que están relacionados con la edad. Una colestasis neonatal transitoria, la aparición de esplenomegalia y/o hepatomegalia pueden preceder en años a los síntomas neurológicos. Pacientes y métodos: Presentamos los 6 casos diagnosticados en nuestra unidad en los últimos 20 años. Se han revisado las manifestaciones clínicas, los hallazgos neurorradiológicos (RM) y el análisis molecular de todos ellos. Resultados: Todos se presentaron antes de los 6 años y 5 casos tuvieron afectación hepática y/o colestasis en el periodo neonatal. En 2 casos se detectó ascitis en el periodo prenatal. La presencia de esplenomegalia se objetivó en 5 casos. En todos los casos se detectaron mutaciones en el gen NPC1. Conclusión: Es importante el conocimiento de esta enfermedad y la identificación de los síntomas clínicos precoces para poder diagnosticarla precozmente, lo que conllevaría a un tratamiento adecuado, pudiendo evitar procedimientos innecesarios. Por otra parte es importante asesorar adecuadamente a las familias y proporcionar un consejo genético (AU)

Introduction: Niemann-Pick type C is a lysosomal storage disorder caused by a defect in intracellular trafficking of cholesterol. It is a rare disease, usually caused by mutations in NPC1 gene, but in some cases by mutations in NPC2 gene. Usually it is present in the paediatric age with a great variability of clinical manifestations. This disease leads to neurological degeneration with various age-related symptoms. Transient neonatal cholestasis, the appearance of splenomegaly and/or hepatomegaly may occur years before the neurological symptoms. Patients and methods: We report 6 cases diagnosed in our unit in the last 20 years. We reviewed the clinical manifestations, neuroradiological findings (MRI) and molecular analysis of all of them. Results: The disease began before 6 years of age and 5 cases had liver dysfunction and cholestasis in the neonatal period. Ascites was detected in 2 cases in prenatal period. Five cases have or had splenomegaly. Mutations in NPC1 gene were detected in all of them. Conclusions: It is important to understand this disease and the identification of early clinical symptoms to make an early diagnosis, leading to appropriate treatment and avoiding unnecessary tests. Moreover, it is important to suitably advise families and provide them with genetic counseling (AU)

Síndromes de Pearson y de Kearns-Sayre: dos enfermedades mitocondriales multisistémicas, debidas a deleciones en el ADN mitocondrial / Pearson and Kearns-Sayre syndromes: two multisystem mitochondrial diseases caused by mitochondrial DNA deletions

El síndrome de Pearson (SP) y el síndrome de Kearns-Sayre (SKS) son enfermedades mitocondriales multisistémicas con diferente fenotipo, causadas por deleciones en el ADN mitocondrial (ADN mt).Objetivo: Describir las manifestaciones clínicas y los hallazgos neurorradiológicos, bioquímicos y genético-moleculares de ambos síndromes, con objeto de difundir su conocimiento entre los pediatras. Pacientes y métodos: Se han estudiado retrospectivamente 6 pacientes con SKS y 3 con SP inicial, dos de los cuales evolucionaron a SKS. Resultados: La edad de inicio de los síntomas fue inferior en los SP. Los síntomas más precoces fueron los hematológicos (anemia), seguidos de los renales (Fanconi) y digestivos (insuficiencia pancreática), y de forma más tardía se presenta la afectación ocular, endocrinológica, cardiológica y neurológica. Cuatro pacientes precisaron implantación de marcapasos. Seis casos presentaron alteraciones cerebrales y/o del tronco del encéfalo en la resonancia magnética. Se observó hiperlactatorraquia, hiperproteinorraquia y descenso de ácido fólico en el líquido cefalorraquídeo. La mitad de los SKS presentaron fibras musculares rojo-rasgadas y fibras citocromo C oxidasa negativas. En ocho pacientes se detectó una deleción única del ADN mt. Conclusiones: 1) Las diferencias más acusadas entre el SP y el SKS fueron la edad de comienzo y las manifestaciones iniciales. Los síntomas en la evolución, así como los hallazgos bioquímicos, neurorradiológicos y genéticos, fueron similares. 2) Las enfermedades mitocondriales deberían incluirse en el diagnóstico diferencial del síndrome de Fanconi, el déficit de la hormona del crecimiento y los trastornos de la conducción cardiaca, especialmente en los casos con afectación multiorgánica. El diagnóstico se confirma por la presencia de una gran deleción en el ADN mt (AU)

Kearns-Sayre (KSS) and Pearson syndromes are both multisystem mitochondrial diseases whose underlying genetic defect is a single large-scale mitochondrial DNA (mt DNA) deletion. Objectives: To describe the clinical spectrum of KSS and PS, with the object of spreading the knowledge of these disease to the pediatricians. Patients and methods: We reviewed the clinical notes of 6 patients diagnosed with KSS and 3 patients initially diagnosed with PS. Results: The age at the onset was lower in PS patients. First appearing symptoms were hematological (anemia), followed by renal (Fanconi) and digestive involvement (pancreatic insufficiency). Ophthalmological, endocrinological, cardiological and neurological symptoms were manifested at later stages. Four patients required pacemaker implantation. Six showed cerebral and/or brain stem involvement in MRI. CSF analysis showed increased levels of both lactic acid and proteins where as folate levels were diminished. Half of the KSS patients showed ragged-red fibers and COX negative fibers in their skeletal muscle. A large-scale mt DNA deletion was found in eight patients. Conclusions: 1. The most remarkable differences between PS and KSS were the age at presentation and the initial clinical symptoms; symptoms during evolution, and biochemical, neuroradiological and genetic findings were similar in both disorders,2. Mitochondrial diseases should be included in the differential diagnosis of the Fanconi syndrome, growth hormone deficiency and cardiac conduction disorders. A single large scale mt DNA is essential to confirm the diagnosis (AU)

PDH E1ß deficiency with novel mutations in two patients with Leigh syndrome.

Most cases of pyruvate dehydrogenase complex (PDHc) deficiency are attributable to mutations in the PDHA1 gene which encodes the E(1)α subunit, with few cases of mutations in the genes for E(3), E3BP (E(3) binding protein), E(2) and E(1)-phosphatase being reported. Only seven patients with deficiency of the E(1)ß subunit have been described, with mutations in the PDHB gene in six of them. Clinically they presented with a non-specific encephalomyopathy. We report two patients with new mutations in PDHB and Leigh syndrome. Patient 1 was a boy with neonatal onset of hyperlactataemia, corpus callosum hypoplasia and a convulsive encephalopathy. After neurological deterioration, he died at age 5 months. Autopsy revealed the characteristic features of Leigh syndrome. Patient 2, also a boy, presented a milder clinical course. First symptoms were noticed at age 16 months with muscular hypotonia, lactic acidosis and recurrent episodes of somnolence and transient tetraparesis. MRI revealed bilateral signal hyperintensities in the globus pallidus, midbrain and crura cerebri. PDHc and E(1) activities were deficient in fibroblasts in patient 1; in patient 2 PDHc deficiency was found in skeletal muscle. Mutations in PDHA1 were excluded. Sequencing of PDHB revealed a homozygous point mutation (c.302T>C), causing a predicted amino acid change (p.M101T) in patient 1. Patient 2 is compound heterozygote for mutations c.301A>G (p.M101V) and c.313G>A (p.R105Q). All three mutations appear to destabilize the E(1) enzyme with a decrease of both E(1)α and E(1)ß subunits in immunoblot analysis. To our knowledge, these patients with novel PDHB mutations are the first reported with Leigh syndrome.

Pathogenic mutations in the 5' untranslated region of BCS1L mRNA in mitochondrial complex III deficiency.

Mutations in the assembly chaperone BCS1L constitute a major cause of mitochondrial complex III deficiency. We studied the presence of BCS1L mutations in a complex III-deficient patient with metabolic acidosis, liver failure, and tubulopathy. A previously reported mutation, p.R56X, was identified in one BCS1L allele, and two novel heterozygous mutations, g.1181A>G and g.1164C>G, were detected in the second allele. The g.1181A>G mutation generated an alternative splicing site in the BCS1L transcript, causing a 19-nucleotides deletion in its 5'UTR region. Decreased BCS1L mRNA and protein levels, and a respiratory chain complex III assembly impairment, determine a pathogenic role for the novel BCS1L mutations.

Déficit del cofactor molibdeno como causa de encefalopatía epiléptica precoz / Molybdenum cofactor deficiency as a cause of early epileptic encephalopathy

No disponible