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1.
Cell ; 187(14): 3585-3601.e22, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38821050

RESUMO

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.


Assuntos
Dolicóis , Dolicóis/metabolismo , Dolicóis/biossíntese , Humanos , Glicosilação , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/genética , Masculino , Mutação de Sentido Incorreto , Feminino
3.
Cell ; 169(3): 442-456.e18, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431245

RESUMO

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.


Assuntos
Antineoplásicos/metabolismo , Escherichia coli/metabolismo , Fluoruracila/metabolismo , Microbioma Gastrointestinal , Animais , Autofagia , Caenorhabditis elegans , Morte Celular , Neoplasias Colorretais/tratamento farmacológico , Dieta , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Modelos Animais , Pentosiltransferases/genética
4.
J Biol Chem ; : 107875, 2024 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-39395802

RESUMO

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

5.
Hum Mol Genet ; 31(15): 2571-2581, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35262690

RESUMO

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.


Assuntos
Retículo Endoplasmático , Bicamadas Lipídicas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Células HeLa , Humanos , Bicamadas Lipídicas/análise , Bicamadas Lipídicas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qc-SNARE/análise , Proteínas Qc-SNARE/metabolismo , Ubiquitinas/metabolismo
6.
Am J Hum Genet ; 108(11): 2130-2144, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34653363

RESUMO

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Genes Dominantes , Hexosiltransferases/genética , Proteínas de Membrana/genética , Doenças Musculoesqueléticas/genética , Doenças do Sistema Nervoso/genética , Adolescente , Adulto , Sequência de Aminoácidos , Domínio Catalítico , Pré-Escolar , Feminino , Heterozigoto , Hexosiltransferases/química , Humanos , Masculino , Proteínas de Membrana/química , Pessoa de Meia-Idade , Linhagem , Homologia de Sequência de Aminoácidos
7.
J Inherit Metab Dis ; 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39038845

RESUMO

ALDH7A1 deficiency is an epileptic encephalopathy whose seizures respond to treatment with supraphysiological doses of pyridoxine. It arises as a result of damaging variants in ALDH7A1, a gene in the lysine catabolism pathway. α-Aminoadipic semialdehyde (α-AASA) and Δ1-piperideine-6-carboxylate (P6C), which accumulate because of the block in the lysine pathway, are diagnostic biomarkers for this disorder. Recently, it has been reported that 6-oxo-pipecolic acid (6-oxo-PIP) also accumulates in the urine, CSF and plasma of ALDH7A1-deficient individuals and that, given its improved stability, it may be a more suitable biomarker for this disorder. This study measured 6-oxo-PIP in urine from a cohort of 30 patients where α-AASA was elevated and showed that it was above the normal range in all those above 6 months of age. However, 6-oxo-PIP levels were within the normal range in 33% of the patients below 6 months of age. Levels increased with age and correlated with a decrease in α-AASA levels. Longitudinal analysis of urine samples from ALDH7A1-deficient patients who were on a lysine restricted diet whilst receiving supraphysiological doses of pyridoxine showed that levels of 6-oxo-PIP remained elevated whilst α-AASA decreased. Similar to α-AASA, we found that elevated urinary excretion of 6-oxo-PIP can also occur in individuals with molybdenum cofactor deficiency. This study demonstrates that urinary 6-oxo-PIP may not be a suitable biomarker for ALDH7A1 deficiency in neonates. However, further studies are needed to understand the biochemistry leading to its accumulation and its potential long-term side effects.

8.
J Inherit Metab Dis ; 46(6): 1170-1185, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37540500

RESUMO

CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.


Assuntos
Di-Hidro-Orotase , Proteínas , Humanos , Di-Hidro-Orotase/química , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Mutação de Sentido Incorreto , Uridina
9.
Hum Genet ; 141(7): 1279-1286, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35182234

RESUMO

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.


Assuntos
Proteínas de Transporte de Cátions , Síndromes de Imunodeficiência , Transtornos Linfoproliferativos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Magnésio/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
10.
Ann Neurol ; 86(2): 225-240, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31187503

RESUMO

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.


Assuntos
Mutação/genética , Polineuropatias/tratamento farmacológico , Polineuropatias/genética , Piridoxal Quinase/genética , Fosfato de Piridoxal/administração & dosagem , Complexo Vitamínico B/administração & dosagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Suplementos Nutricionais , Feminino , Redes Reguladoras de Genes/genética , Humanos , Masculino , Resultado do Tratamento
11.
J Inherit Metab Dis ; 42(4): 629-646, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30671974

RESUMO

Vitamin B6 is present in our diet in many forms, however, only pyridoxal 5'-phosphate (PLP) can function as a cofactor for enzymes. The intestine absorbs nonphosphorylated B6 vitamers, which are converted by specific enzymes to the active PLP form. The role of PLP is enabled by its reactive aldehyde group. Pathways reliant on PLP include amino acid and neurotransmitter metabolism, folate and 1-carbon metabolism, protein and polyamine synthesis, carbohydrate and lipid metabolism, mitochondrial function and erythropoiesis. Besides the role of PLP as a cofactor B6 vitamers also play other cellular roles, for example, as antioxidants, modifying expression and action of steroid hormone receptors, affecting immune function, as chaperones and as an antagonist of Adenosine-5'-triphosphate (ATP) at P2 purinoceptors. Because of the vital role of PLP in neurotransmitter metabolism, particularly synthesis of the inhibitory transmitter γ-aminobutyric acid, it is not surprising that various inborn errors leading to PLP deficiency manifest as B6 -responsive epilepsy, usually of early onset. This includes pyridox(am)ine phosphate oxidase deficiency (a disorder affecting PLP synthesis and recycling), disorders affecting PLP import into the brain (hypophosphatasia and glycosylphosphatidylinositol anchor synthesis defects), a disorder of an intracellular PLP-binding protein (PLPBP, previously named PROSC) and disorders where metabolites accumulate that inactivate PLP, for example, ALDH7A1 deficiency and hyperprolinaemia type II. Patients with these disorders can show rapid control of seizures in response to either pyridoxine and/or PLP with a lifelong dependency on supraphysiological vitamin B6 supply. The clinical and biochemical features of disorders leading to B6 -responsive seizures and the treatment of these disorders are described in this review.


Assuntos
Epilepsia/etiologia , Deficiência de Vitamina B 6/complicações , Vitamina B 6/metabolismo , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Humanos , Erros Inatos do Metabolismo/metabolismo , Prolina/sangue , Fosfato de Piridoxal/uso terapêutico , Piridoxaminafosfato Oxidase/deficiência , Piridoxina/uso terapêutico
12.
Anal Chem ; 89(17): 8892-8900, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28782931

RESUMO

We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B6, affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Epilepsia/diagnóstico , Piridoxaminafosfato Oxidase/metabolismo , Espectrometria de Massas em Tandem/métodos , Adolescente , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Criança , Pré-Escolar , Teste em Amostras de Sangue Seco , Epilepsia/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Masculino , Fosfato de Piridoxal/sangue , Piridoxamina/análogos & derivados , Piridoxamina/sangue , Curva ROC , Vitamina B 6/química , Vitamina B 6/metabolismo , Vitamina B 6/uso terapêutico , Adulto Jovem
13.
Sci Adv ; 10(5): eadk8173, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38295165

RESUMO

The tendency for proteins to form aggregates is an inherent part of every proteome and arises from the self-assembly of short protein segments called aggregation-prone regions (APRs). While posttranslational modifications (PTMs) have been implicated in modulating protein aggregation, their direct role in APRs remains poorly understood. In this study, we used a combination of proteome-wide computational analyses and biophysical techniques to investigate the potential involvement of PTMs in aggregation regulation. Our findings reveal that while most PTM types are disfavored near APRs, N-glycosylation is enriched and evolutionarily selected, especially in proteins prone to misfolding. Experimentally, we show that N-glycosylation inhibits the aggregation of peptides in vitro through steric hindrance. Moreover, mining existing proteomics data, we find that the loss of N-glycans at the flanks of APRs leads to specific protein aggregation in Neuro2a cells. Our findings indicate that, among its many molecular functions, N-glycosylation directly prevents protein aggregation in higher eukaryotes.


Assuntos
Agregados Proteicos , Proteoma , Glicosilação , Proteoma/química , Peptídeos/química , Processamento de Proteína Pós-Traducional
14.
bioRxiv ; 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38948797

RESUMO

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

15.
Biochim Biophys Acta Gen Subj ; 1865(11): 129976, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34358634

RESUMO

Congenital Disorders of Glycosylation (CDG) are an expanding and complex group of rare genetic disorders caused by defects in the glycosylation of proteins and lipids. The genetic spectrum of CDG is extremely broad with mutations in over 140 genes leading to a wide variety of symptoms ranging from mild to severe and life-threatening. There has been an expansion in the genetic complexity of CDG in recent years. More specifically several examples of alternate phenotypes in recessive forms of CDG and new types of CDG following an autosomal dominant inheritance pattern have been identified. In addition, novel genetic mechanisms such as expansion repeats have been reported and several already known disorders have been classified as CDG as their pathophysiology was better elucidated. Furthermore, we consider the future and outlook of CDG genetics, with a focus on exploration of the non-coding genome using whole genome sequencing, RNA-seq and multi-omics technology.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Humanos
16.
JIMD Rep ; 58(1): 122-128, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33728255

RESUMO

Recently, a disorder caused by the heterozygous de novo c.1267C>T (p.R423*) substitution in SLC37A4 has been described. This causes mislocalization of the glucose-6-phosphate transporter to the Golgi leading to a congenital disorder of glycosylation type II (SLC37A4-CDG). Only one patient has been reported showing liver disease that improved with age and mild dysmorphism. Here we report the second patient with a type II CDG caused by the same heterozygous de novo c.1267C>T (p.R423*) mutation thereby confirming the pathogenicity of this variant and expanding the clinical picture with type 1 diabetes, severe scoliosis, and membranoproliferative glomerulonephritis. Additional clinical and biochemical data provide further insight into the mechanism and prognosis of SLC37A4-CDG.

17.
J Pharm Pharmacol ; 69(4): 480-488, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28220480

RESUMO

OBJECTIVES: To assess the pyridoxal 5'-phosphate (PLP) content and stability of extemporaneous PLP liquids prepared from dietary supplements used for the treatment of vitamin B6 -dependent epilepsy. METHODS: Pyridoxal 5'-phosphate liquids were prepared in accordance with the guidelines given to patients from marketed 50 mg PLP dietary capsules and tablets. The PLP content and its stability were evaluated under conditions resembling the clinical setting using reverse phase HPLC and mass spectrometry. KEY FINDINGS: Pyridoxal 5'-phosphate content in most of the extemporaneously prepared liquids from dietary supplements was found to be different from the expected amount (~16-60 mg). Most of these PLP extemporaneous liquids were stable at room temperature (protected from light) after 24 h but unstable after 4 h when exposed to light. A key photodegradation product of PLP in water was confirmed as 4-pyridoxic acid 5'-phosphate (PAP). CONCLUSION: Pyridoxal 5'-phosphate tablets from Solgar® were found to be the most reliable product for the preparation of extemporaneous PLP liquids. This work highlighted the difference between the marketed PLP dietary supplements quality and the importance of proper storage of aqueous PLP. There is a need to develop pharmaceutical forms of PLP that ensure dose accuracy and avoid potentially unsafe impurities with the aim of enhancing safety and compliance.


Assuntos
Epilepsia , Fosfato de Piridoxal/química , Fosfato de Piridoxal/normas , Controle de Qualidade , Complexo Vitamínico B/química , Complexo Vitamínico B/normas , Suplementos Nutricionais/normas , Formas de Dosagem , Estabilidade de Medicamentos , Armazenamento de Medicamentos/normas , Epilepsia/tratamento farmacológico , Soluções Farmacêuticas , Fotólise , Fosfato de Piridoxal/uso terapêutico , Complexo Vitamínico B/uso terapêutico
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