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Objective Birth prevalence of Cobalamin (Cbl) C or D defects in Portugal is an estimated 1:85,000, one of the highest worldwide. We compared the genotype/phenotype of patients identified with CblC or CblD before and after the implementation of expanded newborn screening. Methods Twenty-five Portuguese CblC/D patients, 14 symptomatic and 11 identified through screening, were diagnosed using gas chromatography or tandem mass spectrometry. Molecular characterization was performed through the study of MMACHC and MMADHC genes. Results The most common MMACHC mutation, c.271dupA, was present in 100% of MMACHC alleles of all CblC screened patients, in contrast with the 61% identified before expanded newborn screening. All studied cases (except one, who presented a CblD deficiency) presented a CblC defect. More CblC late-onset patients were diagnosed before the introduction of newborn screening than in the post newborn screening era, probably because some early onset patients died without a definitive diagnosis. Conclusion The molecular data found in this cohort contribute to the improvement of screening and diagnosis of Cbl defects and would enable a confirmatory diagnosis of these patients, reducing the need for complex, costly, laborious, and time-consuming biochemical/enzymatic tests.

Title Deficiencia de glicerolcinasa: una causa metabolica de retraso global del desarrollo.

Item does not contain fulltext Creatine transporter deficiency is an X-linked disorder caused by mutations in the SLC6A8 gene. Currently, 38 pathogenic, including 15 missense variants, are reported. In this study, we report 33 novel, including 6 missense variants. To classify all known missense variants, we transfected creatine deficient fibroblasts with the SLC6A8 ORF containing one of the unique variants and tested their ability to restore creatine uptake. This resulted in the definitive classification of 2 non-disease associated and 19 pathogenic variants of which 3 have residual activity. Furthermore, we report the development and validation of a novel DHPLC method for the detection of heterozygous SLC6A8 variants. The method was validated by analysis of DNAs that in total contained 67 unique variants of which 66 could be detected. Therefore, this rapid screening method may prove valuable for the analysis of large cohorts of females with mild intellectual disability of unknown etiology, since in this group heterozygous SLC6A8 mutations may be detected. DHPLC proved also to be important for the detection of somatic mosaicism in mothers of patients who have a pathogenic mutation in SLC6A8. All variants reported in the present and previous studies are included in the Leiden Open Source Variant Database (LOVD) of SLC6A8 (www.LOVD.nl/SLC6A8).

A new gas chromatography-mass spectrometry method for routine quantification of urine creatine and guanidinoacetic acid (GAA) has been developed to provide a fast, reliable and inexpensive metabolic screening. Our method uses a two-step derivatization procedure which involves a reaction with hexafluoroacetylacetone followed by a reaction with mono-trimethylsilyltrifluoroacetamide. The standard curves showed linearity over a range of 43-4269 micromol/l for GAA and 38-7325 micromol/l for creatine, which covers the range of GAA and creatine normally found in urine. The lower detection limit is 1.54 micromol/l for GAA and 1.22 micromol/l for creatine, whereas the lower quantification limit is 5.04 micromol/l for GAA and 4.19 micromol/l for creatine. This method was also employed to establish reference values for GAA and creatine in healthy infants, children and adolescents based on the analysis of 169 urine samples. Although no sex differences were observed, normal GAA urinary levels and creatine excretion are distinct in age-related subgroups. We identified a statistically significant age difference in two major groups for GAA (children under 4 years, 18-159 micromol/mmol creatinine; and subjects of 5-16 years, 18-130 micromol/mmol creatinine) whereas three groups were discriminated for creatine (children under 4 years, 0.04-1.51 mmol/mmol creatinine; subjects of 5-11 years, 0.04-1.07 mmol/mmol creatinine; and subjects of 12-16 years, 0.04-0.56 mmol/mmol creatinine).

In this study, measurements of guanidinoacetate (GAA) and creatine (Cr) in urine, plasma, and cerebrospinal fluid (CSF) were performed using stable isotope dilution gas chromatography-mass spectrometry. Both compounds were analyzed in a single analysis. Reference values were established for GAA and Cr. These values were age dependent. No differences with gender were observed. Eight guanidinoacetate methyltransferase (GAMT) deficient patients and eight creatine transporter SLC6A8 deficient patients were investigated. In urine, plasma, and CSF of GAMT deficient patients increased levels of GAA are present. The SLC6A8 deficient patients all show increased creatine/creatinine (Cr/Crn) ratio in urine demonstrating the importance of the Cr/Crn ratio as a pathognomonic marker of the SLC6A8 deficiency.

Succinyl-coenzyme A synthase is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinate and adenosine triphosphate (ATP) from succinyl-coenzyme A and adenosine diphosphate (ADP) in the tricarboxylic acid cycle. This enzyme is made up of α and β subunits encoded by SUCLG1 and SUCLA2, respectively. We present a child with severe muscular hypotonia, dystonia, failure to thrive, sensorineural deafness, and dysmorphism. Metabolic investigations disclosed hyperlactacidemia, moderate urinary excretion of methylmalonic acid, and elevated levels of C4-dicarboxylic carnitine in blood. We identified a novel homozygous p.M329V in SUCLA2. In cultured cells, the p.M329V resulted in a reduced amount of the SUCLA2 protein, impaired production of mitochondrial ATP, and enhanced production of reactive oxygen species, which was partially reduced by using 5-aminoimidazole-4-carboxamide ribonucleotide in the culture medium. Expanding the array of SUCLA2 mutations, we suggested that reactive oxygen species scavengers are likely to impact on disease prognosis. This work was partially supported by the National Institute of Health, INSA, (to LV). CeN’s work was performed as part of her PhD thesis under the rules of the Portuguese Foundation for Science and Technology (SFRH/BD/ 45247/2008). The Transnational Cooperation between FCT/CAPES 2013/2014 partially supported, as well, the work of both researchers (Project 6818).

Item does not contain fulltext BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype-genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

Cerebral creatine transporter deficiency, attributable to mutations in the SLC6A8 gene, causes X-linked mental retardation, language delay, epilepsy, and autistic features. In contrast with creatine synthesis defects, the vast majority of patients with SLC6A8 deficiency do not respond to treatment. We describe a Portuguese family with a mutation (c.456C>T; p.Gln486X) in the SL6CA8 gene: two adult monozygotic twin brothers, with psychomotor delay and severe speech impairment. The family also includes their maternal half-sister with psychomotor retardation, predominantly in language, and their mentally retarded mother. This family illustrates the remarkable phenotypic variability in this condition. Investigation of creatine metabolism is mandatory in patients with developmental delay of unknown etiology, to detect this condition.

L-2-hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a neurometabolic disorder caused by the toxic accumulation of high concentration of L-2-hydroxyglutaric acid in plasma and cerebrospinal fluid. Distinct mutations on the L2HGDH gene have been associated with the clinical and biochemical phenotype. Here we present three novel mutations (Gln197X, Gly211Val and c.540+1 G>A), which increase the present deleterious collection of L2HGDH gene up to 35 mutations that we have compiled in this study. In addition, we used the haplotypic information based on polymorphic markers to demonstrate the common origin of Gly57Arg harboring chromosomes.