Disorder Of Glycosylation Type Ia Research Articles

Mutations in PMM2 gene in four unrelated Spanish families with polycystic kidney disease and hyperinsulinemic hypoglycemia

Objectives Hyperinsulinemic hypoglucemia (HH) is characterized by a dysregulation of insulin secretion from pancreatic β cells. Congenital hyperinsulinism has been associated with specific genes in monogenic forms and also with other diseases with a yet unknown genetic cause. In 2017, Rubio Cabezas etal. described the association of HH and autosomal recessive polycystic kidney disease (ARPKD) with a promoter mutation in the PMM2 gene. They found that all the patients carried a promoter mutation (c-167G>T) in PMM2, either homozygous or in trans with a second PMM2 coding mutation. Methods We performed the study of the PMM2 gene in six patients from four unrelated families, previously diagnosed with ARPKD and HH. Results All these patients had in common the heterozygous variant c-167G>T in the promoter region for PMM2. Additionally, each patient carried a compound heterozygote for a second missense mutation in this gene (p.Arg141His, p.Asp148Asn or p.Phe157Ser), previously reported as pathogenic for congenital disorder of glycosylation type Ia, with an autosomal recessive inheritance pattern. Unlike the previous published article, two of our patients showed altered type 1 pattern and one of them with rectal bleeding that could be a sign of PMM2-congenital disorders of glycosylation. Conclusion We propose the study of this gene when carrying out the diagnosis of patients with HH, especially in the neonatal period and when a recessive polycystic kidney disease without alterations in PKDH1 is diagnosed.

In vitro treatment of congenital disorder of glycosylation type Ia using PLGA nanoparticles loaded with GDP‑Man.

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N‑glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose‑6‑P to mannose‑1‑P, which results in low concentration levels of guanosine 5'‑diphospho‑D‑mannose (GDP‑Man), a nucleotide‑activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP‑Man‑loaded poly (D,L‑lactide‑co‑glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG‑Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α‑mannosidase, β‑glucoronidase and β‑galactosidase in defective and normal fibroblasts. GDP‑Man (30 µg/ml GDP‑Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α‑mannosidase and β‑galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of β‑glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG‑Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two‑dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP‑Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.

Metabolic liver diseases presenting with neonatal cholestasis: at the crossroad between old and new paradigms.

Metabolic liver diseases (MLD) are an important group of disorders presenting with neonatal cholestasis (NC). The spectrum of liver involvement is wide and the presumptive diagnosis is traditionally based on clinical and laboratory findings. Recently, next-generation sequencing (NGS) panels have emerged as an appealing tool to diagnose neonatal/infantile cholestatic disorders. The aim of this study was to identify clinical phenotypes of liver injury and contribute to find a diagnostic methodology that integrates new molecular diagnostic tools. We retrospectively analyzed the clinical and biochemical features of 16 patients with MLD and NC. Patients were categorized into three groups: A-NC with liver failure (N = 8): tyrosinemia type I (n = 2), classic galactosemia (n = 5), mitochondrial DNA depletion syndrome (n = 1); B-NC evolving with chronic liver disease (N = 5): argininemia (n = 2); mitochondrial cytopathy (n = 1); congenital disorders of glycosylation type Ia (n = 1); Zellweger syndrome (n = 1); and C-transient NC (N = 3): Niemann-Pick type C (n = 2), citrullinemia type II(n = 1).Conclusion: MLD presenting with NC can be categorized into three main clinical phenotypes of liver injury. We highlight transient NC as a clue for MLD that must be pursued. New molecular diagnostic tools can play a key role, but application criteria must be established to make them cost-effective. What is Known: • Metabolic liver diseases are an important group of disorders presenting with neonatal cholestasis. • The diagnostic approach is challenging and traditionally based on clinical and laboratory findings. Next-generation sequencing is a recent and rapidly developing tool in pediatric hepatology. What is New: • We provide a liver-targeted characterization of metabolic liver diseases presenting with neonatal cholestasis, categorizing them into three clinical phenotypes that may narrow the diagnostic possibilities. • A clinical decision-making algorithm is proposed, in which the NGS technology is integrated.

Inborn errors of metabolism associated with hyperglycaemic ketoacidosis and diabetes mellitus: narrative review.

Inborn errors of metabolism (IEM) are heterogeneous group of disorders that might present in the clinics or emergency departments in different phenotypes, and one of these is a diabetes scenario. Diabetes is the most common endocrine disorder among children. The mechanism of how IEM could lead to diabetes is unclear; however, the postulated pathogenesis consists of three mechanisms: 1) accumulation of toxic substance in the gland, ruining structure and normal functionality, 2) disturbing energy availability required for hormone synthesis and 3) defect of complex molecules. The differential diagnosis of IEM associated with hyperglycaemic ketoacidosis and diabetes include: organic acidemias specifically propionic acidemia, methylmalonic acidemia, isovaleric acidemia, hereditary hemochromatosis, aceruloplasminemia, holocarboxylase synthetase deficiency, β-ketothiolase deficiency and finally, cystinosis, Rogers syndrome (thiamine-responsive megaloblastic anaemia) and congenital disorders of glycosylation type Ia. Clinical approach will help in ready diagnosis and treatment for IEM disorders in early detection of diabetes. In this review, we will discuss the differential diagnosis, clinical features and diagnostic approaches of IEM presenting as hyperglycaemic ketoacidosis and diabetes.

Magnetic Resonance Imaging of Malformations of Midbrain-Hindbrain.

We aim to review the magnetic resonance imaging appearance of malformations of midbrain and hindbrain. These can be classified as predominantly cerebellar malformations, combined cerebellar and brain stem malformations, and predominantly brain stem malformations. The diagnostic criteria for the majority of these morphological malformations are based on neuroimaging findings. The predominantly cerebellar malformations include predominantly vermian hypoplasia seen in Dandy-Walker malformation and rhombencephalosynapsis, global cerebellar hypoplasia reported in lissencephaly and microlissencephaly, and unilateral cerebellar hypoplasia seen in PHACES, vanishing cerebellum, and cerebellar cleft. Cerebellar dysplasias are seen in Chudley-McCullough syndrome, associated with LAMA1 mutations and GPR56 mutations; Lhermitte-Duclos disease; and focal cerebellar dysplasias. Cerebellar hyperplasias are seen in megalencephaly-related syndromes and hemimegalencephaly with ipsilateral cerebellomegaly. Cerebellar and brain stem malformations include tubulinopathies, Joubert syndrome, cobblestone malformations, pontocerebellar hypoplasias, and congenital disorders of glycosylation type Ia. Predominantly brain stem malformations include congenital innervation dysgenesis syndrome, pontine tegmental cap dysplasia, diencephalic-mesencephalic junction dysplasia, disconnection syndrome, and pontine clefts.

The Effects of PMM2-CDG-Causing Mutations on the Folding, Activity, and Stability of the PMM2 Protein

Congenital disorder of glycosylation type Ia (PMM2-CDG), the most common form of CDG, is caused by mutations in the PMM2 gene that reduce phosphomannomutase 2 (PMM2) activity. No curative treatment is available. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The effects of the mutations on PMM2/Pmm2 stability, oligomerization, and enzyme activity were explored in an optimized bacterial system. The mutant proteins were associated with an enzymatic activity of up to 47.3% as compared with wild type (WT). Stability analysis performed using differential scanning fluorimetry and a bacterial transcription-translation-coupled system allowed the identification of several destabilizing mutations (p.V44A, p.D65Y, p.R123Q, p.R141H, p.R162W, p.F207S, p.T237M, p.C241S). Exclusion chromatography identified one mutation, p.P113L, that affected dimer interaction. Expression analysis of the p.V44A, p.D65Y, p.R162W, and p.T237M mutations in a eukaryotic expression system under permissive folding conditions showed the possibility of recovering their associated PMM2 activity. Together, the results suggest that some loss-of-function mutations detected in PMM2-CDG patients could be destabilizing, and therefore PMM2 activity could be, in certain cases, rescuable via the use of synergetic proteostasis modulators and/or chaperones.

Nuclear-mitochondrial proteins: too much to process?

This scientific commentary refers to ‘ PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia’, by Jobling et al. (doi:10.1093/brain/awv057). In the not too distant past, non-progressive focal neurological deficits emerging during child development were all labelled as ‘cerebral palsy’, and assumed to be caused by an insult during pregnancy or in the perinatal period. This view has changed dramatically over the last two decades, in large part as a result of advances in structural brain imaging, which failed to identify the characteristic patterns of hypoxic-ischaemic brain injury or perinatal infection in every case. Metabolic and genetic studies subsequently unravelled a diverse and unexpected range of mechanisms responsible for these ‘static’ neurological deficits, and the pace has accelerated with the application of exome sequencing (Srivastava et al. , 2014). Patients with non-progressive cerebellar ataxia have been particularly difficult to explain. The number of known causes has remained limited, and includes extreme rarities such as congenital disorder of glycosylation type Ia, caused by phosphomannomutase 2 deficiency (Boddaert et al. , 2010). However, the differential diagnosis and mechanisms responsible for this phenotype have now broadened with the findings of Jobling et al. (2015), described in this issue of Brain . Initially studying a family from the Bekaa valley in north east Lebanon, Jobling et al. looked for regions of shared homozygosity using microsatellite markers in 12 individuals with non-progressive ataxia, spasticity, and intellectual disability (Delague et al. , 2002). All shared a 12.1-cM homozygous region on chromosome 9q34-qter between markers D9S67 and D9S312. Exome sequencing identified homozygous mutations in two genes within the mapped candidate region: PMPCA and CAMSAP1 , neither previously associated with disease. These findings presented a …

N-Glycan Abnormalities in Children with Galactosemia

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

Hypertrophic Cardiomyopathy With Cardiac Rupture and Tamponade Caused by Congenital Disorder of Glycosylation Type Ia

Hypertrophic cardiomyopathy (HCM) is a rare presenting feature of congenital disorder of glycosylation type Ia (CDG-Ia). We report two female siblings with CDG-Ia and cardiomyopathy. Patient no. 1 died at 12 days of age from cardiac rupture and tamponade, which has not previously been reported in CDG-Ia. The second patient died at 2 months of age from HCM. The severe cardiac manifestations seen in our patients emphasize the importance of early cardiac assessment in all patients with CDG-Ia.

Potent, Selective, and Orally Available Benzoisothiazolone Phosphomannose Isomerase Inhibitors as Probes for Congenital Disorder of Glycosylation Ia

We report the discovery and validation of a series of benzoisothiazolones as potent inhibitors of phosphomannose isomerase (PMI), an enzyme that converts mannose-6-phosphate (Man-6-P) into fructose-6-phosphate (Fru-6-P) and, more importantly, competes with phosphomannomutase 2 (PMM2) for Man-6-P, diverting this substrate from critical protein glycosylation events. In congenital disorder of glycosylation type Ia, PMM2 activity is compromised; thus, PMI inhibition is a potential strategy for the development of therapeutics. High-throughput screening (HTS) and subsequent chemical optimization led to the identification of a novel class of benzoisothiazolones as potent PMI inhibitors having little or no PMM2 inhibition. Two complementary synthetic routes were developed, enabling the critical structural requirements for activity to be determined, and the compounds were subsequently profiled in biochemical and cellular assays to assess efficacy. The most promising compounds were also profiled for bioavailability parameters, including metabolic stability, plasma stability, and permeability. The pharmacokinetic profile of a representative of this series (compound 19; ML089) was also assessed, demonstrating the potential of this series for in vivo efficacy when dosed orally in disease models.

Amniotic fluid α-fetoprotein microheterogeneity in the prenatal diagnosis of congenital disorders of glycosylation type Ia

Congenital disorders of glycosylation are a group of clinically and biochemically diverse defects. The current screening method (based on analysis of transferrin), which is used postnatally for the most frequent types, is however not suitable for prenatal diagnosis. The aim of the study was to investigate whether alterations in the microheterogeneity of α-fetoprotein would provide more reliable results. During the 14th-19th weeks of gestation, 140 amniotic fluid samples were obtained by amniocentesis and tested for fetal developmental abnormalities. α-Fetoprotein was analyzed using isoelectric focusing on Immobiline DryPlate pH 4-7, rehydrated in urea (8 mol/L), and molecular forms of the glycoprotein were detected by immunofixation and silver staining. A difference in the relative proportion of individual α-fetoprotein bands (particularly increase of band II density) was found in a case where a congenital disorder of glycosylation was diagnosed postnatally, and in two other samples from pregnancies which resulted in termination, without further examination. Our potential for further testing is limited; thus far, no other congenital disorders of glycosylation-positive samples have been available. Verification of our results in another laboratory with the exclusion of several potentially pertinent variables is advisable.

Congenital disorder of glycosylation type Ia: Heterogeneity in the clinical presentation from multivisceral failure to hyperinsulinaemic hypoglycaemia as leading symptoms in three infants with phosphomannomutase deficiency

We describe three patients with congenital disorder of glycosylation (CDG) type Ia, all of whom had persistent hyperinsulinaemic hypoglycaemia responding to diazoxide therapy as a common feature. The first patient, an infant girl, presented with recurrent vomiting, failure to thrive, liver impairment, hypothyroidism and a pericardial effusion. The second patient, also female, had a milder disease with single organ involvement, presenting as isolated hyperinsulinaemic hypoglycaemia, not associated with any cognitive impairment. The third patient, a boy presented with multi-organ manifestations including congenital hypothyroidism, persistent hyperinsulinaemic hypoglycaemia, coagulopathy, olivopontocerebellar hypoplasia and recurrent pancreatitis. All three patients had a type 1 serum transferrin isoform pattern, and were subsequently found to have low phosphomannomutase activity, confirming the diagnosis of CDG type Ia. Our findings emphasize that CDG should be considered as a differential diagnosis in patients with persistent hyperinsulinaemic hypoglycaemia and that it may even occasionally be the leading symptom in CDG Ia.

Conotruncal heart defects in three patients with congenital disorder of glycosylation type Ia (CDG Ia)

Congenital heart defects (CHDs) are a group of structural abnormalities of the heart that have a combined incidence of approximately 1% in humans. It is estimated that 4–5% of CHDs...

Functional analysis of three splicing mutations identified in the PMM2 gene: Toward a new therapy for congenital disorder of glycosylation type Ia

The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N-glycosylation. The most prevalent form of CDG-type Ia-is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256-1G>C and c.640-9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640-15479C>T). Cell-based splicing assays strongly suggest that all these are disease-causing splicing mutations. The c.256-1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640-9T>G mutation was found responsible for the activation of a cryptic intronic splice-site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine-rich (SR) proteins. Finally, the deep intronic change c.640-15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation-specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen.

Congenital disorder of glycosylation type Ia in a Malaysian family: Clinical outcome and description of a novel PMM2 mutation

There are few reports of congenital disorders of glycosylation (CDGs) in the Asian population, although they have been reported worldwide. We identified a Malaysian infant female at 2 days of life with CDG type Ia. The diagnosis was suspected on the basis of inverted nipples and abnormal fat distribution. She had cerebellar hypoplasia and developed coagulopathy, hypothyroidism and severe pericardial effusion and died at 7 months of life. The diagnosis was supported by abnormal serum transferrin isoform pattern that showed elevated levels of the disialotransferrin isoform and trace levels of the asialotransferrin isoform. Enzyme testing of peripheral leukocytes showed decreased level of phosphomannomutase (PMM) activity (0.6 nmol/min per mg protein, normal range 1.6-6.2) and a normal level of phosphomannose isomerase activity (19 nmol/min per mg protein, normal range 12-25), indicating a diagnosis of CDG type Ia. Mutation study of the PMM2 gene showed the patient was heterozygous for both the common p.R141H (c.422T>A) mutation and a novel sequence change in exon 7, c.618C>A. The latter change is predicted to result in the replacement of the highly conserved phenylalanine residue at position 206 with a leucine residue (p.F206L) and occurs in the same codon as the previously reported p.F206S mutation. Analysis of 100 control chromosomes has shown that the p.F206L sequence change is not present, making it highly likely that this change is functionally important. To the best of our knowledge, this is the first report of CDG in the Malay population. Prenatal diagnosis was successfully performed in a subsequent pregnancy for this family.

CDG Type Ia and Congenital Cytomegalovirus Infection: Two Coexisting Conditions

Congenital disorders of glycosylation are a heterogeneous group of disorders with multisystemic involvement. The most common form is phosphomannomutase deficiency or congenital disorders of glycosylation type Ia with an autosomal recessive inheritance and incidence estimated at 1/20000-1/50000 live born. Congenital disorders of glycosylation Ia can manifest as severe multisystemic disease of infancy or milder disorder with only neurological problems including ataxia, hypotonia, and psychomotor retardation. The brain pathological findings in congenital disorders of glycosylation type Ia patients corroborate with cerebellar dysfunction. Usually the most affected part is the anterior lobe of the vermis. Microscopic analysis demonstrates the prominent Purkinje cell loss and subtotal loss of the external and internal granule cell layers. The authors present clinical and pathological picture of a 4-month-old girl with congenital disorders of glycosylation type Ia, additionally complicated by congenital cytomegalovirus infection. The diagnosis was confirmed by low phosphomannomutase activity in patient's fibroblasts and mutations on both alleles of phosphomannomutase 2 gene.

Congenital disorder of glycosylation type Ia: a non-progressive encephalopathy associated with multisystemic involvement

Congenital disorders of glycosylation (CDG) are a group of severe, autosomal recessive, multisystemic diseases, characterized by abnormal glycosylation of glycoproteins and glycolipids1. The first disease of this group was reported in 19802. Since then, at least 16 entities were recognized and were classified in two groups (CDG-Ia to IL and CDG-IIa to IId). […] Congenital disorder of glycosylation type Ia: a non-progressive encephalopathy associated with multisystemic involvement

Pericardial and abdominal fluid accumulation in Congenital Disorder of Glycosylation type Ia

The association of fetal hydrops with Congenital Disorders of Glycosylation (CDG) has been reported previously. Pericardial fluid accumulation and ascites were also observed in a few young patients with CDG type Ia. Here we describe the clinical and biochemical features in three children developing life-threatening extravascular fluid accumulation. All patients carried severe PMM2 mutations comparable to the earlier reported patients with fetal hydrops. One patient was successfully treated with a pericardial–pleural shunt placement. Pericardial fluid accumulation and generalized oedema resolved temporarily in the other two children on regular albumin infusions and the use of diuretics. Sequential abdominal punctures were unsuccessful in the treatment of the extensive ascites production. The use of non-steroid anti-inflammatory agents and the application of high dose steroids had no clinical effect. Severe extravascular fluid accumulation progressed to decompensation and death. Biochemical investigations of the abdominal fluid and pericardial fluid demonstrated a high extracellular protein concentration, increased cytokine concentrations and an abnormal transferrin isoelectric focusing pattern characteristic of CDG type I. Our results are consistent with a local activation of the cytokine pathways and subsequent protein transport through the endothelial surface to the extravascular space. Normal glycosylation of cell surface proteins is essential for the normal fluid balance and protein transport through the pericardial and peritoneal membrane. Future therapeutic efforts should be directed to inhibit the abnormal immune response and excessive protein transport in this life-threatening complication of CDG syndrome.

Different neuroradiological findings during two stroke-like episodes in a patient with a congenital disorder of glycosylation type Ia

Congenital disorders of glycosylation type Ia (CDG-Ia) are the most common type of CDG and are characterized by liver dysfunction, coagulation disorders, mental retardation, hypotonia, cerebellar dysfunction, polyneuropathy, seizures, and stroke-like episodes. Stroke-like episodes occur in 40–55% of cases, but their etiology is not fully understood. Although it has been stated that an epileptic process may cause the stroke-like episodes, there is no clear evidence of ischemic stroke. Here, we describe two stroke-like episodes in a patient with CDG. We performed radiological studies during each episode and obtained two distinct magnetic resonance imaging (MRI) findings: one revealed an ischemic stroke, and the other demonstrated marked edema followed by focal necrosis. This is the first direct evidence of ischemic stroke, and we report that another process may affect the etiology in the same patient.

The first case of congenital disorders of glycosylation type Ia in Russia

The first case of congenital disorders of glycosylation type Ia in Russia