ABSTRACT
BACKGROUND: Propionic acidemia (PA) is an inherited disorder caused by deficiency of propionyl CoA carboxylase. Most patients with this disorder are diagnosed during the neonatal period because of severe metabolic acidosis and hyperammonemia. Patients are required to undergo blood and urine analysis at least 3 to 4 times per year, depending on age and metabolic control. METHODS: We designed a prospective study in which we investigated the results from blood and urinary samples collected monthly in filter paper from 10 PA patients followed in a single metabolic reference center from January 2015 to September 2017. The aim of this study was to evaluate the usefulness of filter paper samples in the follow-up of the PA patients. RESULTS: During the follow-up period, 163 dried blood spot (DBS) and 119 urine dried spot samples were analyzed and compared with 160 plasma and 103 liquid urine specimens; 64 specimens of plasma were analyzed for odd-numbered long-chain fatty acids (OLCFAs). A total of 40 metabolic crises, 18 of them with hyperammonemia were documented. We observed a strong correlation between the filter paper and the urine/plasma samples for the main PA parameters both in stable metabolic conditions as well as in acute decompensations. Also, there was a strong correlation between OLCFAs measured in plasma and quantification of odd number acylcarnitines in DBS. CONCLUSIONS: We conclude that filter paper blood and urinary samples can be used for the follow-up of the patients with PA, correctly reflecting their metabolic situation.
ABSTRACT
Cardiac alterations (hypertrophic/dilated cardiomyopathy, and rhythm alterations) are one of the major causes of mortality and morbidity in propionic acidemia (PA), caused by the deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC), involved in the catabolism of branched-chain amino acids, cholesterol, and odd-chain fatty acids. Impaired mitochondrial oxidative phosphorylation has been documented in heart biopsies of PA patients, as well as in the hypomorphic Pcca-/-(A138T) mouse model, in the latter correlating with increased oxidative damage and elevated expression of cardiac dysfunction biomarkers atrial and brain natriuretic peptides (ANP and BNP) and beta-myosin heavy chain (ß-MHC). Here we characterize the cardiac phenotype in the PA mouse model by histological and echocardiography studies and identify a series of upregulated cardiac-enriched microRNAs (miRNAs) in the PA mouse heart, some of them also altered as circulating miRNAs in PA patients' plasma samples. In PA mice hearts, we show alterations in signaling pathways regulated by the identified miRNAs, which could be contributing to cardiac remodeling and dysfunction; notably, an activation of the mammalian target of rapamycin (mTOR) pathway and a decrease in autophagy, which are reverted by rapamycin treatment. In vitro studies in HL-1 cardiomyocytes indicate that propionate, the major toxic metabolite accumulating in the disease, triggers the increase in expression levels of miRNAs, BNP, and ß-MHC, concomitant with an increase in reactive oxygen species. Our results highlight miRNAs and signaling alterations in the PCC-deficient heart which may contribute to the development of PA-associated cardiomyopathy and provide a basis to identify new targets for therapeutic intervention.
Subject(s)
Cardiomyopathies/genetics , MicroRNAs/genetics , Propionic Acidemia/genetics , Animals , Cardiomyopathies/complications , Cardiomyopathies/physiopathology , Cell Line , Echocardiography , Humans , Mice , Propionic Acidemia/complications , Propionic Acidemia/physiopathology , Signal TransductionABSTRACT
Congenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group's experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system's workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system's use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this condition.
ABSTRACT
A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742Câ¯>â¯G (p.Pro581Arg) and c.2368Câ¯>â¯T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery.
Subject(s)
Hyperglycinemia, Nonketotic/genetics , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mutation/genetics , Cell Differentiation/genetics , Cell Differentiation/physiology , Humans , Infant, Newborn , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Male , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407â¯fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency.
Subject(s)
Homozygote , Induced Pluripotent Stem Cells , Methylmalonyl-CoA Decarboxylase , Mutation , Propionic Acidemia , Cell Line , Humans , Induced Pluripotent Stem Cells/enzymology , Induced Pluripotent Stem Cells/pathology , Kruppel-Like Factor 4 , Methylmalonyl-CoA Decarboxylase/genetics , Methylmalonyl-CoA Decarboxylase/metabolism , Propionic Acidemia/enzymology , Propionic Acidemia/genetics , Propionic Acidemia/pathologyABSTRACT
OBJECTIVE: Phosphomannomutase deficiency (PMM2 congenital disorder of glycosylation [PMM2-CDG]) causes cerebellar syndrome and strokelike episodes (SLEs). SLEs are also described in patients with gain-of-function mutations in the CaV2.1 channel, for which acetazolamide therapy is suggested. Impairment in N-glycosylation of CaV2.1 promotes gain-of-function effects and may participate in cerebellar syndrome in PMM2-CDG. AZATAX was designed to establish whether acetazolamide is safe and improves cerebellar syndrome in PMM2-CDG. METHODS: A clinical trial included PMM2-CDG patients, with a 6-month first-phase single acetazolamide therapy group, followed by a randomized 5-week withdrawal phase. Safety was assessed. The primary outcome measure was improvement in the International Cooperative Ataxia Rating Scale (ICARS). Other measures were the Nijmegen Pediatric CDG Rating Scale (NPCRS), a syllable repetition test (PATA test), and cognitive scores. RESULTS: Twenty-four patients (mean age = 12.3 ± 4.5 years) were included, showing no serious adverse events. Thirteen patients required dose adjustment due to low bicarbonate or asthenia. There were improvements on ICARS (34.9 ± 23.2 vs 40.7 ± 24.8, effect size = 1.48, 95% confidence interval [CI] = 4.0-7.6, p < 0.001), detected at 6 weeks in 18 patients among the 20 responders, on NPCRS (95% CI = 0.3-1.6, p = 0.013) and on the PATA test (95% CI = 0.5-3.0, p = 0.006). Acetazolamide improved prothrombin time, factor X, and antithrombin. Clinical severity, epilepsy, and lipodystrophy predicted greater response. The randomized withdrawal phase showed ICARS worsening in the withdrawal group (effect size = 1.46, 95% CI = 2.65-7.52, p = 0.001). INTERPRETATION: AZATAX is the first clinical trial of PMM2-CDG. Acetazolamide is well tolerated and effective for motor cerebellar syndrome. Its ability to prevent SLEs and its long-term effects on kidney function should be addressed in future studies. Ann Neurol 2019;85:740-751.
Subject(s)
Acetazolamide/therapeutic use , Carbonic Anhydrase Inhibitors/therapeutic use , Cerebellar Diseases/diagnosis , Cerebellar Diseases/drug therapy , Congenital Disorders of Glycosylation/diagnosis , Congenital Disorders of Glycosylation/drug therapy , Phosphotransferases (Phosphomutases)/deficiency , Acetazolamide/pharmacology , Adolescent , Carbonic Anhydrase Inhibitors/pharmacology , Cerebellar Diseases/genetics , Child , Child, Preschool , Congenital Disorders of Glycosylation/genetics , Female , Glycosylation/drug effects , Humans , Male , Phosphotransferases (Phosphomutases)/genetics , Single-Blind Method , Treatment Outcome , Young AdultABSTRACT
The present work describes the value of genetic analysis as a confirmatory measure following the detection of suspected inborn errors of metabolism in the Spanish newborn mass spectrometry screening program. One hundred and forty-one consecutive DNA samples were analyzed by next-generation sequencing using a customized exome sequencing panel. When required, the Illumina extended clinical exome panel was used, as was Sanger sequencing or transcriptional profiling. Biochemical tests were used to confirm the results of the genetic analysis. Using the customized panel, the metabolic disease suspected in 83 newborns (59%) was confirmed. In three further cases, two monoallelic variants were detected for two genes involved in the same biochemical pathway. In the remainder, either a single variant or no variant was identified. Given the persistent absence of biochemical alterations, carrier status was assigned in 39 cases. False positives were recorded for 11. In five cases in which the biochemical pattern was persistently altered, further genetic analysis allowed the detection of two variants affecting the function of BCAT2, ACSF3, and DNAJC12, as well as a second, deep intronic variant in ETFDH or PTS. The present results suggest that genetic analysis using extended next-generation sequencing panels can be used as a confirmatory test for suspected inborn errors of metabolism detected in newborn screening programs. Biochemical tests can be very helpful when a diagnosis is unclear. In summary, simultaneous genomic and metabolomic analyses can increase the number of inborn errors of metabolism that can be confirmed following suggestive newborn screening results.
Subject(s)
Genetic Testing , Lipid Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/genetics , Neonatal Screening , Exome/genetics , High-Throughput Nucleotide Sequencing , Humans , Infant, Newborn , Lipid Metabolism, Inborn Errors/diagnosis , Lipid Metabolism, Inborn Errors/epidemiology , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/epidemiology , Mutation/genetics , Spain/epidemiology , Exome SequencingABSTRACT
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.
Subject(s)
Congenital Disorders of Glycosylation/diagnosis , Congenital Disorders of Glycosylation/genetics , Phosphotransferases (Phosphomutases)/genetics , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , SpainABSTRACT
PURPOSE: We report a patient with a human cationic amino acid transporter 2 (CAT-2) defect discovered due to a suspected arginase 1 deficiency observed in newborn screening (NBS). METHODS: A NBS sample was analyzed using tandem mass spectrometry. Screen results were confirmed by plasma and urine amino acid quantification. Molecular diagnosis was done using clinical exome sequencing. Dimethylated arginines were determined by HPLC and nitrate/nitrite levels by a colorimetric assay. The metabolomic profile was analyzed using 1D nuclear magnetic resonance spectroscopy. RESULTS: A Spanish boy of nonconsanguineous parents had high arginine levels in a NBS blood sample. Plasma and urinary cationic amino acids were high. Arginase enzyme activity in erythrocytes was normal and no pathogenic mutations were identified in the ARG1 gene. Massive parallel sequencing detected two loss-of-function mutations in the SLC7A2 gene. Currently, the child receives a protein-controlled diet of 1.2 g/kg/day with protein-and amino-acid free infant formula, 30 g/day, and is asymptomatic. CONCLUSION: We identified a novel defect in human CAT-2 due to biallelic pathogenic variants in the SLC7A2 gene. The characteristic biochemical profile includes high plasma and urine arginine, ornithine, and lysine levels. NBS centers should know of this disorder since it can be detected in arginase 1 deficiency screening.
Subject(s)
Amino Acid Transport Systems, Basic/genetics , Cationic Amino Acid Transporter 2/deficiency , Metabolic Diseases/genetics , Arginase/genetics , Diet, Protein-Restricted , Humans , Hyperargininemia/genetics , Infant, Newborn , Male , Metabolic Diseases/diet therapy , Mutation , Neonatal ScreeningABSTRACT
INTRODUCTION: Phosphomannomutase-2 deficiency (PMM2-CDG) is associated with a recognisable facial pattern. There are no early severity predictors for this disorder and no phenotype-genotype correlation. We performed a detailed dysmorphology evaluation to describe facial gestalt and its changes over time, to train digital recognition facial analysis tools and to identify early severity predictors. METHODS: Paediatric PMM2-CDG patients were evaluated and compared with controls. A computer-assisted recognition tool was trained. Through the evaluation of dysmorphic features (DFs), a simple categorisation was created and correlated with clinical and neurological scores, and neuroimaging. RESULTS: Dysmorphology analysis of 31 patients (4-19 years of age) identified eight major DFs (strabismus, upslanted eyes, long fingers, lipodystrophy, wide mouth, inverted nipples, long philtrum and joint laxity) with predictive value using receiver operating characteristic (ROC) curveanalysis (p<0.001). Dysmorphology categorisation using lipodystrophy and inverted nipples was employed to divide patients into three groups that are correlated with global clinical and neurological scores, and neuroimaging (p=0.005, 0.003 and 0.002, respectively). After Face2Gene training, PMM2-CDG patients were correctly identified at different ages. CONCLUSIONS: PMM2-CDG patients' DFs are consistent and inform about clinical severity when no clear phenotype-genotype correlation is known. We propose a classification of DFs into major and minor with diagnostic risk implications. At present, Face2Gene is useful to suggest PMM2-CDG. Regarding the prognostic value of DFs, we elaborated a simple severity dysmorphology categorisation with predictive value, and we identified five major DFs associated with clinical severity. Both dysmorphology and digital analysis may help physicians to diagnose PMM2-CDG sooner.
Subject(s)
Congenital Disorders of Glycosylation/diagnosis , Congenital Disorders of Glycosylation/genetics , Facies , Genetic Association Studies , Genetic Predisposition to Disease , Phenotype , Phosphotransferases (Phosphomutases)/deficiency , Adolescent , Child , Child, Preschool , Female , Genetic Testing , Humans , Male , Phosphotransferases (Phosphomutases)/genetics , ROC Curve , Spain , Young AdultABSTRACT
Propionic acidemia (PA) is caused by mutations in the PCCA and PCCB genes, encoding α and ß subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Up to date, >200 pathogenic mutations have been identified, mostly missense defects. Genetic analysis in PA patients referred to the laboratory for the past 15â¯years identified 20 novel variants in the PCCA gene and 14 in the PCCB gene. 21 missense variants were predicted as probably disease-causing by different bioinformatics algorithms. Structural analysis in the available 3D model of the PCC enzyme indicated potential instability for most of them. Functional analysis in a eukaryotic system confirmed the pathogenic effect for the missense variants and for one amino acid deletion, as they all exhibited reduced or null PCC activity and protein levels compared to wild-type constructs. PCCB variants p.E168del, p.Q58P and p.I460T resulted in medium-high protein levels and no activity. Variants p.R230C and p.C712S in PCCA, and p.G188A, p.R272W and p.H534R in PCCB retained both partial PCC activity and medium-high protein levels. Available patients-derived fibroblasts carriers of some of these mutations were grown at 28⯰C or 37⯰C and a slight increase in PCC activity or protein could be detected in some cases at the folding-permissive conditions. Examination of available clinical data showed correlation of the results of the functional analysis with disease severity for most mutations, with some notable exceptions, confirming the notion that the final phenotypic outcome in PA is not easily predicted.
Subject(s)
Genetic Predisposition to Disease , Methylmalonyl-CoA Decarboxylase/genetics , Propionic Acidemia/genetics , Structure-Activity Relationship , Adolescent , Adult , Child , Child, Preschool , Female , Genetic Association Studies , Genotype , Humans , Infant , Infant, Newborn , Male , Methylmalonyl-CoA Decarboxylase/chemistry , Mitochondria/enzymology , Mitochondria/genetics , Mutation, Missense/genetics , Neonatal Screening , Propionic Acidemia/pathology , Protein Conformation , Protein Folding , Young AdultABSTRACT
BACKGROUND: Cellular cobalamin defects are a locus and allelic heterogeneous disorder. The gold standard for coming to genetic diagnoses of cobalamin defects has for some time been gene-by-gene Sanger sequencing of individual DNA fragments. Enzymatic and cellular methods are employed before such sequencing to help in the selection of the gene defects to be sought, but this is time-consuming and laborious. Furthermore some cases remain undiagnosed because no biochemical methods have been available to test for cobalamin absorption and transport defects. RESULTS: This paper reports the use of massive parallel sequencing of DNA (exome analysis) for the accurate and rapid genetic diagnosis of cobalamin-related defects in a cohort of affected patients. The method was first validated in an initial cohort with different cobalamin defects. Mendelian segregation, the frequency of mutations, and the comprehensive structural and functional analysis of gene variants, identified disease-causing mutations in 12 genes involved in the absorption and synthesis of active cofactors of vitamin B12 (22 cases), and in the non-cobalamin metabolism-related genes ACSF3 (in four biochemically misdiagnosed patients) and SUCLA2 (in one patient with an unusual presentation). We have identified thirteen new variants all classified as pathogenic according to the ACGM recommendation but four were classified as variant likely pathogenic in MUT and SUCLA2. Functional and structural analysis provided evidences to classify them as pathogenic variants. CONCLUSIONS: The present findings suggest that the technology used is sufficiently sensitive and specific, and the results it provides sufficiently reproducible, to recommend its use as a second-tier test after the biochemical detection of cobalamin disorder markers in the first days of life. However, for accurate diagnoses to be made, biochemical and functional tests that allow comprehensive clinical phenotyping are also needed.
Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , Homocystinuria/genetics , Vitamin B 12 Deficiency/genetics , Coenzyme A Ligases/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Mutation/genetics , Succinate-CoA Ligases/genetics , Vitamin B 12/metabolism , Vitamin B 12 Deficiency/metabolismABSTRACT
Stroke-like episodes (SLE) occur in phosphomannomutase deficiency (PMM2-CDG), and may complicate the course of channelopathies related to Familial Hemiplegic Migraine (FHM) caused by mutations in CACNA1A (encoding CaV2.1 channel). The underlying pathomechanisms are unknown. We analyze clinical variables to detect risk factors for SLE in a series of 43 PMM2-CDG patients. We explore the hypothesis of abnormal CaV2.1 function due to aberrant N-glycosylation as a potential novel pathomechanism of SLE and ataxia in PMM2-CDG by using whole-cell patch-clamp, N-glycosylation blockade and mutagenesis. Nine SLE were identified. Neuroimages showed no signs of stroke. Comparison of characteristics between SLE positive versus negative patients' group showed no differences. Acute and chronic phenotypes of patients with PMM2-CDG or CACNA1A channelopathies show similarities. Hypoglycosylation of both CaV2.1 subunits (α1A and α2α) induced gain-of-function effects on channel gating that mirrored those reported for pathogenic CACNA1A mutations linked to FHM and ataxia. Unoccupied N-glycosylation site N283 at α1A contributes to a gain-of-function by lessening CaV2.1 inactivation. Hypoglycosylation of the α2δ subunit also participates in the gain-of-function effect by promoting voltage-dependent opening of the CaV2.1 channel. CaV2.1 hypoglycosylation may cause ataxia and SLEs in PMM2-CDG patients. Aberrant CaV2.1 N-glycosylation as a novel pathomechanism in PMM2-CDG opens new therapeutic possibilities.
Subject(s)
Cerebellar Diseases/complications , Channelopathies/complications , Phosphotransferases (Phosphomutases)/deficiency , Stroke/complications , Adolescent , Amino Acid Sequence , Calcium Channels/genetics , Cerebellar Diseases/diagnostic imaging , Channelopathies/diagnostic imaging , Child , Child, Preschool , Electroencephalography , Female , Glycosylation , HEK293 Cells , Humans , Ion Channel Gating/drug effects , Magnetic Resonance Imaging , Male , Mutation/genetics , Phosphotransferases (Phosphomutases)/chemistry , Phosphotransferases (Phosphomutases)/metabolism , Stroke/diagnostic imaging , Tunicamycin/pharmacologyABSTRACT
The original supplementary information included with this article contained several minor errors. Corrected Supplementary Information accompanies this corrigendum.
ABSTRACT
Human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia carrying mutations in the PCCA gene: c.1899+4_1899+7delAGTA; p.(Cys616_Val633del) and c.1430--?_1643+?del; p.(Gly477Glufs*9). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.
Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/pathology , Methylmalonyl-CoA Decarboxylase/genetics , Propionic Acidemia/pathology , Base Sequence , Cell Line , Humans , Kruppel-Like Factor 4 , Reproducibility of ResultsABSTRACT
miRNome expression profiling was performed in a mouse model of propionic acidemia (PA) and in patients' plasma samples to investigate the role of miRNAs in the pathophysiology of the disease and to identify novel biomarkers and therapeutic targets. PA is a potentially lethal neurometabolic disease with patients developing neurological deficits and cardiomyopathy in the long-term, among other complications. In the PA mouse liver we identified 14 significantly dysregulated miRNAs. Three selected miRNAs, miR-34a-5p, miR-338-3p and miR-350, were found upregulated in brain and heart tissues. Predicted targets involved in apoptosis, stress-signaling and mitochondrial function, were inversely found down-regulated. Functional analysis with miRNA mimics in cellular models confirmed these findings. miRNA profiling in plasma samples from neonatal PA patients and age-matched control individuals identified a set of differentially expressed miRNAs, several were coincident with those identified in the PA mouse, among them miR-34a-5p and miR-338-3p. These two miRNAs were also found dysregulated in childhood and adult PA patients' cohorts. Taken together, the results reveal miRNA signatures in PA useful to identify potential biomarkers, to refine the understanding of the molecular mechanisms of this rare disease and, eventually, to improve the management of patients.
Subject(s)
Gene Expression Regulation , MicroRNAs/analysis , MicroRNAs/blood , Propionic Acidemia/pathology , Propionic Acidemia/physiopathology , Animals , Brain/pathology , Disease Models, Animal , Gene Expression Profiling , Humans , Infant, Newborn , Liver/pathology , Mice , Myocardium/pathology , Plasma/chemistryABSTRACT
Pathogenic mutations in DPAGT1 are manifested as two possible phenotypes: congenital disorder of glycosylation DPAGT1-CDG (also known as CDG-Ij), and limb-girdle congenital myasthenic syndrome (CMS) with tubular aggregates. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosamine phosphotransferase (GPT), the protein encoded by DPAGT1, is an endoplasmic reticulum (ER)-resident protein involved in an initial step in the N-glycosylation pathway. The aim of the present study was to examine the effect of six variants in DPAGT1 detected in patients with DPAGT1-CDG, and the role of endoplasmic reticulum stress, as part of the search for therapeutic strategies to use against DPAGT1-CDG. The effect of the six mutations, i.e., c.358C>A (p.Leu120Met), c.791T>G (p.Val264Gly), c.901C>T (p.Arg301Cys), c.902G>A (p.Arg301His), c.1154T>G (p.Leu385Arg), and of the novel mutation c.329T>C (p.Phe110Ser), were examined via the analysis of DPAGT1 transcriptional profiles and GTP levels in patient-derived fibroblasts. In addition, the transient expression of different mutations was analysed in COS-7 cells. The results obtained, together with those of bioinformatic studies, revealed these mutations to affect the splicing process, the stability of GTP, or the ability of this protein to correctly localise in the ER membrane. The unfolded protein response (UPR; the response to ER stress) was found not to be active in patient-derived fibroblasts, unlike that seen in cells from patients with PMM2-CDG or DPM1-CDG. Even so, the fibroblasts of patients with DPAGT1-CDG seemed to be more sensitive to the stressor tunicamycin. The present work improves our knowledge of DPAGT1-CDG and provides bases for developing tailored splicing and folding therapies.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Endoplasmic Reticulum Stress , Mutation , N-Acetylglucosaminyltransferases/physiology , Animals , COS Cells , Chlorocebus aethiops , Humans , Microscopy, Fluorescence , N-Acetylglucosaminyltransferases/genetics , Polymorphism, Single Nucleotide , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcription, GeneticABSTRACT
Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic ß cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2 We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy.