Jolanta Sykut-Cegielska

SRD5A3 Is Required for Converting Polyprenol to Dolichol and Is Mutated in a Congenital Glycosylation Disorder

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

Multiple phenotypes in phosphoglucomutase 1 deficiency

BACKGROUND Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

Retrospective, multicentric study of 180 children with cytochrome c oxidase deficiency

A retrospective, multicenter study of 180 children with cytochrome c oxidase (COX) deficiency analyzed the clinical features, prognosis, and molecular bases of the COX deficiency. Clinical symptoms including failure to thrive, encephalopathy, hypotony, Leigh syndrome, cardiac involvement, and hepatopathy appeared in most patients early after birth or in early childhood. Two thirds of all children died. Biochemical examination revealed an isolated COX deficiency in 101 children and COX deficiency combined with disturbances of other respiratory chain complexes in 79 children. Blood and cerebrospinal fluid lactate increased in 85% and 81% of examined cases, respectively. Pathogenic mutations in mitochondrial or nuclear DNA were established in 75 patients. Mutations in surfeit locus protein 1 gene (SURF1) were found in 47 children with Leigh syndrome; 2bp deletion 845-846delCT was found in 89% of independent alleles. Mutations in a mitochondrial copper-binding protein (SCO2) gene were found in nine children with encephalomyopathy and/or cardiomyopathy; all of them were homozygotes or heterozygotes for 1541G>A mutation. Different mitochondrial DNA (mtDNA) deletion or depletion were found in nine children, mtDNA mutation 3243A>G in six, mtDNA mutation 8363G>A in two children with Leigh syndrome and mtDNA mutations 8344A>G, and 9205-9206delTA in one child each. COX deficiency represents a heterogeneous group of diseases with unfavorable prognosis. Marked prevalence of two nuclear DNA mutations (845-846delCT in the SURF1 gene and 1541G>A in the SCO2 gene) associated with COX deficiency in a Slavonic population suggests the existence of regional differences in the genetic basis of COX deficiency.

Sepiapterin reductase deficiency: A Treatable Mimic of Cerebral Palsy

Sepiapterin reductase deficiency (SRD) is an under‐recognized levodopa‐responsive disorder. We describe clinical, biochemical, and molecular findings in a cohort of patients with this treatable condition. We aim to improve awareness of the phenotype and available diagnostic and therapeutic strategies to reduce delayed diagnosis or misdiagnosis, optimize management, and improve understanding of pathophysiologic mechanisms.

A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism

Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype with severe, early visual impairment and variable eye malformations, including optic nerve hypoplasia, retinal coloboma, congenital cataract and glaucoma. Some of the symptoms overlapped with the phenotype in other congenital disorders of glycosylation type I subtypes, such as vermis hypoplasia, anaemia, ichtyosiform dermatitis, liver dysfunction and coagulation abnormalities. We recently identified pathogenic mutations in the SRD5A3 gene, encoding steroid 5α-reductase type 3, in a group of patients who presented with this particular phenotype and a common metabolic pattern. Here, we report on the clinical, genetic and metabolic features of 12 patients from nine families with cerebellar ataxia and congenital eye malformations diagnosed with SRD5A3-congenital disorders of glycosylation due to steroid 5α-reductase type 3 defect. This enzyme is necessary for the reduction of polyprenol to dolichol, the lipid anchor for N-glycosylation in the endoplasmic reticulum. Dolichol synthesis is an essential metabolic step in protein glycosylation. The current defect leads to a severely abnormal glycosylation state already in the early phase of the N-glycan biosynthesis pathway in the endoplasmic reticulum. We detected high expression of SRD5A3 in foetal brain tissue, especially in the cerebellum, consistent with the finding of the congenital cerebellar malformations. Based on the overlapping clinical, biochemical and genetic data in this large group of patients with congenital disorders of glycosylation, we define a novel syndrome of cerebellar ataxia associated with congenital eye malformations due to a defect in dolichol metabolism.

Defining the phenotype in an autosomal recessive cutis laxa syndrome with a combined congenital defect of glycosylation

Autosomal recessive cutis laxa is a genetically heterogeneous condition. Its molecular basis is largely unknown. Recently, a combined disorder of N- and O-linked glycosylation was described in children with congenital cutis laxa in association with severe central nervous system involvement, brain migration defects, seizures and hearing loss. We report on seven additional patients with similar clinical features in combination with congenital disorder of glycosylation type IIx. On the basis of phenotype in 10 patients, we define an autosomal recessive cutis laxa syndrome. The patients have a complex phenotype of neonatal cutis laxa, transient feeding intolerance, late closure of the fontanel, characteristic facial features including down-slanting palpebral fissures, short nose and small mouth, and developmental delay. There is a variable degree of the central nervous system involvement and variable systemic presentation. The biochemical analysis using transferrin isoelectric focusing gives false negative results in some of the youngest patients. Analysis of the apolipoprotein C-III isoelectric focusing, however, is diagnostic in all cases.

The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 2: the evolving clinical phenotype

BackgroundThe disease course and long-term outcome of patients with organic acidurias (OAD) and urea cycle disorders (UCD) are incompletely understood.AimsTo evaluate the complex clinical phenotype of OAD and UCD patients at different ages.ResultsAcquired microcephaly and movement disorders were common in OAD and UCD highlighting that the brain is the major organ involved in these diseases. Cardiomyopathy [methylmalonic (MMA) and propionic aciduria (PA)], prolonged QTc interval (PA), optic nerve atrophy [MMA, isovaleric aciduria (IVA)], pancytopenia (PA), and macrocephaly [glutaric aciduria type 1 (GA1)] were exclusively found in OAD patients, whereas hepatic involvement was more frequent in UCD patients, in particular in argininosuccinate lyase (ASL) deficiency. Chronic renal failure was often found in MMA, with highest frequency in mut0 patients. Unexpectedly, chronic renal failure was also observed in adolescent and adult patients with GA1 and ASL deficiency. It had a similar frequency in patients with or without a movement disorder suggesting different pathophysiology. Thirteen patients (classic OAD: 3, UCD: 10) died during the study interval, ten of them during the initial metabolic crisis in the newborn period. Male patients with late-onset ornithine transcarbamylase deficiency were presumably overrepresented in the study population.ConclusionsNeurologic impairment is common in OAD and UCD, whereas the involvement of other organs (heart, liver, kidneys, eyes) follows a disease-specific pattern. The identification of unexpected chronic renal failure in GA1 and ASL deficiency emphasizes the importance of a systematic follow-up in patients with rare diseases.

Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency

Adenylosuccinate lyase (ADSL) catalyzes two steps in purine nucleotide metabolism-the 8th step in the de novo pathway: conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazole carboxamide ribotide (AICAR), and conversion of adenylosuccinate (S-AMP) to adenylate (AMP) in the purine nucleotide cycle. To date, over 50 patients have been reported suffering from ADSL deficiency. We report all seven so far diagnosed Polish patients with this defect. Most of our patients shared intractable seizures and psychomotor retardation since the neonatal period and had biochemical evidence of severe (type I) deficiency. Two patients with type II suffered only from mild/moderate psychomotor retardation and showed a transientvisual contact disturbance. One patient had a fatal neonatal form of ADSL deficiency with lack of spontaneous movement, respiratory failure, severe encephalopathy and intractable seizures. Analysis of the ADSL gene showed that four apparently unrelated patients carried a R426H mutation (two homozygous and two compound heterozygous). With the exception of the latter mutation, a Y114H mutation that had been reported previously, and a novel mutation T242I, all other mutations (including D268H and three novel S23R, D215H and I351T mutations) were found only in single families in single alleles. A search for this disorder should be included in the screening program of all infants with unexplained neonatal seizures, severe infantile epileptic encephalopathy, developmental delay, hypotonia, and/or autistic features.

Ophthalmological abnormalities in children with congenital disorders of glycosylation type I

Background: Children with congenital disorders of glycosylation (CDG) type Ia frequently present with ocular involvement and visual loss. Little is known, however, about the occurrence of ophthalmological abnormalities in other subtypes of CDG syndrome. Methods: We evaluated 45 children sequentially diagnosed with CDG type I for the presence of ocular abnormalities at the time of the diagnosis and during follow-up. We compared the various ophthalmic findings in the different CDG subgroups. Results: Of the 45 patients, 22 had CDG type Ia, nine had CDG type Ic and 14 had a so-far undiagnosed biochemical background (CDG type Ix). We found ocular anomalies in 28 of the 45 children. Three had unique findings, including congenital cataract, retinal coloboma and glaucoma. A few CDG type Ia patients showed a sequential occurrence of symptoms, including retinitis pigmentosa or cataract. Conclusions: Ophthalmic findings are frequent in CDG syndrome involving both the anterior and posterior segment of the eye. The disorder might lead to abnormal development of the lens or the retina, cause diminished vision, alter ocular motility and intraocular pressure. We suggest routine screening and follow-up for ophthalmological anomalies in all children diagnosed with CDG syndrome to provide early treatment and adequate counselling.