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Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Seven subunits of the mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) in humans have been recently described in function and structure. QIL1 (also named MIC13) is a small complex that is crucial for the maintenance and assembling of MICOS. A novel mutation of an essential splice site in the C19orf70 gene encoding QIL1 induces severe mitochondrial encephalopathy, hepatopathy and lactate acidosis consistent with psychomotor retardation. In addition, bilateral kidney stones were observed. Disassembly of MICOS complex subunits displays lack of MIC10-MIC26-MIC27-QIL1 subcomplex, resulting in aberrant cristae structure and a loss of cristae junctions and contact sites. In liver and muscle tissue, the activity of the respiratory chain complexes (OXPHOS) was severely impaired. Defects in MICOS complex do not only affect mitochondrial architecture, but also mitochondrial fusion, metabolic signalling, lipid trafficking and cellular electric homeostasis.

Objective: To demonstrate that mutations in the phosphatidylglycerol remodelling enzyme SERAC1 can cause juvenile-onset complicated hereditary spastic paraplegia (cHSP) clusters, thus adding SERAC1 to the increasing number of complex lipid cHSP genes., Methods: Combined genomic and functional validation studies (whole-exome sequencing, mRNA, cDNA and protein), biomarker investigations (3-methyl-glutaconic acid, filipin staining and phosphatidylglycerols PG34:1/PG36:1), and clinical and imaging phenotyping were performed in six affected subjects from two different branches of a large consanguineous family., Results: 5 of 6 affected subjects shared cHSP as a common disease phenotype. Three subjects presented with juvenile-onset oligosystemic cHSP, still able to walk several miles at age >10-20 years. This benign phenotypic cluster and disease progression is strikingly divergent to the severe infantile phenotype of all SERAC1 cases reported so far. Two family members showed a more multisystemic juvenile-onset cHSP, indicating an intermediate phenotype between the benign oligosystemic cHSP and the classic infantile SERAC1 cluster. The homozygous splice mutation led to loss of the full-length SERAC1 protein and impaired phosphatidylglycerol PG34:1/PG36:1 remodelling. These phosphatidylglycerol changes, however, were milder than in classic infantile-onset SERAC1 cases, which might partially explain the milder SERAC1 phenotype., Conclusions: Our findings add SERAC1 to the increasing list of complex lipid cHSP genes. At the same time they redefine the phenotypic spectrum of SERAC1 deficiency. It is associated not only with the severe infantile-onset 'Methylglutaconic aciduria, Deafness, Encephalopathy, Leigh-like' syndrome (MEGDEL syndrome), but also with oligosystemic juvenile-onset cHSP as part of the now unfolding SERAC1 deficiency spectrum., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The biogenesis of the multi-subunit vacuolar-type H + -ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy., (© 2017 Rujano et al.)

In recent years, many mutations have been identified that affect the biosynthesis of the glycosylphosphatidylinositol anchor, a biomolecule that attaches surface molecules to cell membranes. Here, we present two second-degree cousins with unexplained patterns of seizures. Next-generation sequencing identified the homozygous c.460A>G; p.(R154G) PIGW mutation in both patients. Transfection of the mutated allele into Pigw-defective CHO cells indicated impaired enzymatic activity of the mutated PIGW product. Alkaline phosphatase did not exceed the upper normal range and flow cytometry of CD16, CD24, and CD66c on granulocytes showed subtle changes of the cellular expression of the glycosylphosphatidylinositol-anchored proteins. The patients' phenotype is therefore remarkably different from the phenotype of the only other described individual with PIGW mutations. Patients might therefore be missed when relying on traditional flow cytometry of glycosylphosphatidylinositol-anchored proteins only and we suggest that glycosylphosphatidylinositol-deficiency should be considered even with patients not showing the typical clinical phenotypes. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Aims: Elevated Carbohydrate-deficient transferrin (CDT) levels are used as a biomarker in order to screen for chronic alcohol abuse. Transferrin (Tf) variants can impair methods to measure elevated CDT levels such as high-performance liquid chromatography (HPLC). We present a Tf variant affecting the second glycosylation site of Tf and the complications it causes in diagnosing alcoholism., Methods: A blood sample from a patient with suspected alcohol abuse was analyzed with HPLC, isoelectric focusing, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), immunoprecipitation and SDS-Page. Sanger sequencing of Tf was performed to detect Tf mutations., Results: HPLC, SDS-Page and IEF showed a distinctly increased disialo-Tf fraction while the tetrasialo-Tf fraction was decreased, ESI-TOF-MS confirmed these results. Sanger sequencing revealed the Tf mutation c.1889 A>C, deleting a Tf glycosylations site and thereby causing elevated disialo-Tf levels., Conclusions: Transferrin mutations can severely impair the diagnostics of chronic alcohol abuse by causing false positive results. This has to be considered when CDT screening is used to detect alcoholism., (© The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Objectives: Transferrin variants can hinder the diagnostic process in cases of suspected Congenital disorders of glycosylation which affect N-Glycosylation. In addition they can impair the use of Carbohydrate deficient Transferrin as a biomarker for chronic alcohol abuse, in which Asialo-Transferrin and Disialo-Transferrin are increased. We present a novel transferrin variant as well as an overview of transferrin mutations found at our laboratory., Design and Methods: Blood samples from patients with suspected CDG were analyzed using the standard diagnostic procedures of Isoelectric focusing and High-performance liquid chromatography as well as the additional procedures of neuraminidase digestion of glycans and Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS)., Results: Four known and one previously unreported transferrin variants were identified. Neuraminidase digestion and ESI-TOF MS revealed changes in charge of the transferrin molecules while the glycosylation status was found to be normal., Conclusion: Transferrin variants are pitfalls in the diagnostics of CDG. The found variants change the charge of the transferrin molecule, thus affecting the standard diagnostic procedures. Neuraminidase digestion as well as ESI-TOF MS can identify variants and mutations in a laboratory context., (Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood. These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting. Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence suggesting that thiamine supplementation may rescue TPK enzyme activity. Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK dimerization, which may be a novel mechanism for the disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The analysis of serum transferrin either by high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) is the standard diagnostic procedure in patients with the suspicion of a congenital disorder of glycosylation (CDG). Carbohydrate-deficient transferrin (CDT) is also analysed in monitoring programmes in cases of alcohol abuse. We report a novel transferrin variant that impairs the analysis using conventional methods and propose alternative forms of analysis., Methods: Transferrin samples were analysed using HPLC, immunoprecipitation followed by SDS-PAGE and IEF. Neuraminidase treatment followed by conventional IEF and electrospray ionization time of flight mass spectrometry (ESI-TOF MS) were applied before sequencing of the transferrin gene was performed., Results: The novel transferrin variant E592A, found both in homozygous and heterozygous form, causes an altered charge of the transferrin molecule, which changes the results of IEF and HPLC and mimics an increase in trisialo-transferrin. The change in charge can be detected either by neuraminidase digestion followed by IEF or by ESI-TOF MS., Conclusion: Conventional diagnostic methods for CDG are hindered by the novel transferrin E592A. Neuraminidase treatment followed by IEF and ESI-TOF MS can identify the mutation. The mutation appears to be functionally normal., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases. The deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase, encoded by the human ortholog of ALG1 from yeast, is known as ALG1-CDG (CDG-Ik). The phenotypical, molecular and biochemical analysis of a severely affected ALG1-CDG patient is the focus of this paper. The patient's main symptoms were feeding problems and diarrhea, profound hypoproteinemia with massive ascites, muscular hypertonia, seizures refractory to treatment, recurrent episodes of apnoea, cardiac and hepatic involvement and coagulation anomalies. Compound heterozygosity for the mutations c.1145T>C (M382T) and c.1312C>T (R438W) was detected in the patient's ALG1-coding sequence. In contrast to a previously reported speculation on R438W we confirmed both mutations as disease-causing in ALG1-CDG., (© 2013.)

Congenital disorders of glycosylation (CDG) are caused by enzymatic defects of the formation or processing of lipid-linked oligosaccharides and glycoproteins. Since the majority of proteins is glycosylated, a defect in a singular CDG enzyme leads to a multisytemic disease with secondary malfunction of thousands of proteins. CDG-Ij (DPAGT1-CDG) is caused by a defect of the human DPAGT1 (UDP-GlcNAc: Dolichol Phosphate N-Acetylglucosamine-1-Phosphotransferase), catalyzing the first step of N-linked glycosylation. So far the clinical phenotype of only one CDG-Ij patient has been described. The patient showed severe muscular hypotonia, intractable seizures, developmental delay, mental retardation, microcephaly and exotropia. Molecular studies of this patient revealed the heterozygous mutation c.660A>G (Y170C; paternal) in combination with an uncharacterized splicing defect (maternal). Two further mutations, c.890A>T (I297F) and c.162-8G>A as a splicing defect were detected when analyzing DPAGT1 in two affected siblings of a second family. We report two new patients with the novel homozygous mutation, c.341C>G (A114 G), causing a severe clinical phenotype, characterized by hyperexcitability, intractable seizures, bilateral cataracts, progressive microcephaly and muscular hypotonia. Both our patients died within their first year of life. With the discovery of this novel mutation and a detailed clinical description we extend the clinical features of CDG-Ij in order to improve early detection of this disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G>A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Congenital disorders of glycosylation (CDG) are an expanding group of inherited disorders caused by defects in the N- or O-Glycosylation of proteins and lipids. Several CDG subtypes have been described so far, including CDG type Ih which is caused by a deficiency of the dolichyl-P-Glc:Glc(1)Man(9)GlcNAc(2)-PP-dolichyl alpha1,3-glucosyltransferase (hALG8). The defect leads to an accumulation of Dol-PP-GlcNAc(2)Man(9) and Dol-PP-GlcNAc(2)Man(9)Glc(1) in the endoplasmic reticulum of patients' fibroblasts that can be detected by analyzing the lipid-linked oligosaccharyl intermediates. Five patients with CDG-Ih have been described so far. The clinical presentation of four of these patients was severe with death in early infancy. In this report, we describe two mildly affected siblings with CDG-Ih caused by two novel mutations. While one mutation (c.1434delC) causes a frame shift resulting in a premature termination codon (p.485X), the point mutation of the other allele (c.845C>T, p.A282V) causes an amino acid replacement in a highly conserved region of the hALG8 gene. The two siblings show similar symptoms, including pseudo-gynecomastia, epicanthus, muscular hypotonia, mental retardation and ataxia, expanding the genetic and clinical spectrum of CDG-Ih.

The following study describes the discovery of a new inherited metabolic disorder, dolichol kinase (DK1) deficiency. DK1 is responsible for the final step of the de novo biosynthesis of dolichol phosphate. Dolichol phosphate is involved in several glycosylation reactions, such as N-glycosylation, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and C- and O-mannosylation. We identified four patients who were homozygous for one of two mutations (c.295T-->A [99Cys-->Ser] or c.1322A-->C [441Tyr-->Ser]) in the corresponding hDK1 gene. The residual activity of mutant DK1 was 2%-4% when compared with control cells. The mutated alleles failed to complement the temperature-sensitive phenotype of DK1-deficient yeast cells, whereas the wild-type allele restored the normal growth phenotype. Affected patients present with a very severe clinical phenotype, with death in early infancy. Two of the patients died from dilative cardiomyopathy.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder normally caused by a spontaneous heterozygous mutation in the LMNA gene that codes for the nuclear lamina protein lamin A. Several enzymes are involved in the processing of its precursor, prelamin A, to the mature lamin A. A functional knockout of one of the enzymes involved in prelamin A processing, the zinc metalloprotease ZMPSTE24, causes an even more severe disorder with early neonatal death described as restrictive dermatopathy (RD). This work describes a HGPS patient with a combined defect of a homozygous loss-of-function mutation in the ZMPSTE24 gene and a heterozygous mutation in the LMNA gene that results in a C-terminal elongation of the final lamin A. Whereas the loss of function mutation of ZMPSTE24 normally results in lethal RD, the truncation of LMNA seems to be a salvage alteration alleviating the clinical picture to the HGPS phenotype. The mutations of our patient indicate that farnesylated prelamin A is the deleterious agent leading to the HGPS phenotype, which gives further insights into the pathophysiology of the disorder., (Copyright 2006 Wiley-Liss, Inc.)

The purpose of this study was to identify the biochemical and genetic defect in L-2-hydroxyglutaric aciduria, a neurometabolic disorder characterized by the presence of elevated concentrations of L-2-hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid. Evidence is provided for the existence in rat tissues of a FAD-dependent enzyme catalyzing specifically the oxidation of L-2-hydroxyglutarate to alpha-ketoglutarate. This enzyme is mainly expressed in liver and kidney but also at lower levels in heart, brain, and other tissues. Subcellular fractionation indicates that the liver enzyme is present in mitochondria, where it is bound to membranes. Based on this information, a database search led to the identification of a gene encoding a human hypothetical protein homologous to bacterial FAD-dependent malate dehydrogenases and targeted to mitochondria. The gene encoding this protein, present on chromosome 14q22.1, was found to be in a region homozygous in patients with L-2-hydroxyglutaric aciduria from two consanguineous families. Three mutations that replaced a highly conserved residue (Lys-71-Glu and Glu-176-Asp) or removed exon 9 were identified in homozygous state in patients from three distinct families and were found to cosegregate with the disease. It is concluded that L-2-hydroxyglutarate is normally metabolized to alpha-ketoglutarate in mammalian tissues and that L-2-hydroxyglutaric aciduria is caused by mutations in the gene that most likely encodes L-2-hydroxyglutarate dehydrogenase. The pathological findings observed in this metabolic disorder must therefore be due to a toxic effect of L-2-hydroxyglutarate on the central nervous system.

We describe a new congenital disorder of glycosylation, CDG-If. The patient has severe psychomotor retardation, seizures, failure to thrive, dry skin and scaling with erythroderma, and impaired vision. CDG-If is caused by a defect in the gene MPDU1, the human homologue of hamster Lec35, and is the first disorder to affect the use, rather than the biosynthesis, of donor substrates for lipid-linked oligosaccharides. This leads to the synthesis of incomplete and poorly transferred precursor oligosaccharides lacking both mannose and glucose residues. The patient has a homozygous point mutation (221T-->C, L74S) in a semiconserved amino acid of MPDU1. Chinese hamster ovary Lec35 cells lack a functional Lec35 gene and synthesize truncated lipid-linked oligosaccharides similar to the patient's. They lack glucose and mannose residues donated by Glc-P-Dol and Man-P-Dol. Transfection with the normal human MPDU1 allele nearly completely restores normal glycosylation, whereas transfection with the patient's MPDU1 allele only weakly restores normal glycosylation. This work provides a new clinical picture for another CDG that may involve synthesis of multiple types of glycoconjugates.

Congenital disorder of glycosylation Ic is caused by mutations in the hALG6 gene that encodes an alpha-1,3 glucosyltransferase. This enzyme is required for the addition of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Here we describe the biochemical and molecular analysis of a patient with three mutations in the hALG6 gene. The maternal allele has an intronic G --> A mutation resulting in skipping of exon3 (IVS3 + 5G > A). This produces a nonfunctional enzyme as shown by its inability to restore normal glycosylation in a Saccharomyces cerevisiae strain lacking a functional ALG6. The paternal allele has two mutations. One is a deletion of three bases (895-897delATA) leading to an in-frame deletion of isoleucine 299 (delI299) located in a transmembrane domain. The second mutation on the same allele 911T > C causes a F304S change. When expressed in the ALG6 deficient yeast strain, this allele restores glycosylation but the mRNA is unstable or inefficiently transcribed, contributing to the impaired glycosylation in the patient., (Copyright 2000 Academic Press.)