Progressive myoclonic epilepsy as an expanding phenotype of NGLY1-associated congenital deglycosylation disorder: A case report and review of the literature.

INTRODUCTION: NGLY1-associated congenital disorder of deglycosylation (CDDG1: OMIM #615273) is a rare autosomal recessive disorder caused by a functional impairment of endoplasmic reticulum in degradation of glycoproteins. Neurocognitive dysfunctions have been documented in patients with CDDG1; however, deteriorating phenotypes of affected individuals remain elusive. CASE PRESENTATION: A Japanese boy with delayed psychomotor development showed ataxic movements from age 5 years and myoclonic seizures from age 12 years. Appetite loss, motor and cognitive decline became evident at age 12 years. Electrophysiological studies identified paroxysmal discharges on myoclonic seizure and a giant somatosensory evoked potential. Perampanel was effective for controlling myoclonic seizures. Exome sequencing revealed that the patient carried compound heterozygous variants in NGLY1, NM_018297.4: c.857G > A and c.-17_12del, which were inherited from mother and father, respectively. A literature review confirmed that myoclonic seizures were observed in 28.5% of patients with epilepsy. No other patients had progressive myoclonic epilepsy or cognitive decline in association with loss-of-function variations in NGLY1. CONCLUSION: Our data provides evidence that a group of patients with CDDG1 manifest slowly progressive myoclonic epilepsy and cognitive decline during the long-term clinical course.

Clinical and molecular findings in three Japanese patients with N-acetylneuraminic acid synthetase-congenital disorder of glycosylation (NANS-CDG).

We report clinical and molecular findings in three Japanese patients with N-acetylneuraminic acid synthetase-congenital disorder of glycosylation (NANS-CDG). Patient 1 exhibited a unique constellation of clinical features including marked hydrocephalus, spondyloepimetaphyseal dysplasia (SEMD), and thrombocytopenia which is comparable to that of an infant reported by Faye-Peterson et al., whereas patients 2 and 3 showed Camera-Genevieve type SMED with intellectual/developmental disability which is currently known as the sole disease name for NANS-CDG. Molecular studies revealed a maternally inherited likely pathogenic c.207del:p.(Arg69Serfs*57) variant and a paternally derived likely pathogenic c.979_981dup:p.(Ile327dup) variant in patient 1, a homozygous likely pathogenic c.979_981dup:p.(Ile327dup) variant caused by maternal segmental isodisomy involving NANS in patient 2, and a paternally inherited pathogenic c.133-12T>A variant leading to aberrant splicing and a maternally inherited likely pathogenic c.607T>C:p.(Tyr203His) variant in patient 3 (reference mRNA: NM_018946.4). The results, together with previously reported data, imply that (1) NANS plays an important role in postnatal growth and fetal brain development; (2) SMED is recognizable at birth and shows remarkable postnatal evolution; (3) NANS-CDG is associated with low-normal serum sialic acid, obviously elevated urine N-acetylmannosamine, and normal N- and O-glycosylation of serum proteins; and (4) NANS-CDG is divided into Camera-Genevieve type and more severe Faye-Peterson type.

Mass Spectrometry of Transferrin and Apolipoprotein CIII from Dried Blood Spots for Congenital Disorders of Glycosylation.

Dried blood spot (DBS) is the standard specimen for the newborn screening of inborn errors of metabolism (IEM) by tandem mass spectrometry. Availability of DBS for the mass spectrometric analysis of the diagnostic marker proteins, transferrin (Tf) and apolipoprotein CIII (apoCIII), of congenital disorders of glycosylation (CDG) was examined. Recovery of Tf from DBS was only slightly reduced compared with fresh serum. Although oxidation of the core polypeptides was observed, glycans of Tf and apoCIII were unaffected by storage of DBS in the ambient environment for at least 1 month. The combination of DBS and the triple quadrupole mass spectrometer used for IEM screening was sufficient to characterize the aberrant glycoprofiles of Tf and apoCIII in CDG. DBS or dried serum spot on filter paper can reduce the cost of sample transportation and potentially promote mass spectrometric screening of CDG.

Novel ALG12 variants and hydronephrosis in siblings with impaired N-glycosylation.

BACKGROUND: ALG12-CDG is a rare autosomal recessive type I congenital disorder of glycosylation (CDG) due to pathogenic variants in ALG12 which encodes the dolichyl-P-mannose:Man-7-GlcNAc-2-PP-dolichyl-alpha-6-mannosyltransferase. Thirteen patients from unrelated 11 families have been reported, most of them result in broad multisystem manifestations with clinical variability. It is important to validate abnormal glycosylation to establish causal relationship. CASE REPORT: Here, we report two siblings with novel compound heterozygous variants in ALG12: c.443T>C, p.(Leu148Pro) and c.412_413insCGT, p.(Gln137_Phe138insSer). Both patients showed global developmental delay, microcephaly, hypotonia, failure to thrive, facial dysmorphism, skeletal malformations and coagulation abnormalities, which are common in ALG12-CDG. In addition, one of our patients showed left hydronephrosis, which is a novel clinical feature in ALG12-CDG. Brain MRI showed hypoplasia of cerebrum, brain stem and cerebellar vermis in both patients. N-glycosylation defects of trypsin digested transferrin peptides were revealed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and electrospray ionization MS verified the lack of N-glycans in transferrin. CONCLUSIONS: The present study can add hydronephrosis to phenotypic spectrum of ALG12-CDG. Since the symptoms of ALG12-CDG are quite diverse, the combination of whole-exome sequencing and transferrin glycopeptide analysis with MS, can help diagnosis of ALG12-CDG.

Primary ovarian insufficiency in a female with phosphomannomutase-2 gene (PMM2) mutations for congenital disorder of glycosylation.

Primary ovarian insufficiency (POI) is a highly heterogeneous condition, and its underlying causes remain to be clarified in a large fraction of patients. Congenital disorders of glycosylation (CDG) are multisystem diseases caused by mutations of a number of genes involved in N-glycosylation or O-glycosylation, and the most frequent form is PMM2-CDG (alias, CDG-Ia) resulting from biallelic mutations in PMM2 encoding phosphomannomutase-2 involved in N-glycosylation. Here, we examined a 46,XX Japanese female with syndromic POI accompanied by an undetectable level of serum anti-Müllerian hormone (AMH). Whole exome sequencing identified biallelic pathogenic mutations of PMM2 (a novel c.34G>C:p.(Asp12His) of maternal origin and a recurrent c.310C>G:p.(Leu104Val) of paternal origin) (NM_000303.3), and N-glycosylation studies detected asialotransferrin and disialotransferrin characteristic of PMM2-CDG, in addition to normally glycosylated tetrasialotransferrin. Clinical assessment showed cerebellar hypotrophy, which is a fairly characteristic and highly prevalent feature in PMM2-CDG, together with multiple non-specific features reported in PMM2-CDG such as characteristic face, intellectual disability, skeletal abnormalities, and low blood antithrombin III value. These results including the undetectable level of serum AMH, in conjunction with previously reported findings suggestive of the critical role of glycosylation in oocyte development and function, imply that PMM2-CDG almost invariably leads to POI primarily because of the defective oogenesis and/or oocyte-dependent early folliculogenesis rather than the compromised bioactivity of FSH/LH with defective glycosylation. Thus, it is recommended to examine PMM2 in patients with syndromic POI, especially in those with cerebellar ataxia/hypotrophy.

Decreased sialylation of IgA1 O-glycans associated with pneumococcal hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) in children is usually caused by Shiga-like toxin-producing Escherichia coli, but approximately 5% of cases are caused by invasive pneumococcal infection (P-HUS). Reported herein is the case of a 9-month-old HUS patient with pneumococcal meningitis who needed hemodialysis for 12 days. Decreased sialylation was characterized in both transferrin N-glycans and IgA1 O-glycans, analyzed in the acute phase on mass spectrometry, consistent with S. pneumonia-produced sialidases hydrolyzing both α2,3- and α2,6-linked sialic acids. The method will complement the T-antigen activation test and help to understand the molecular pathology related to P-HUS.

Congenital disorder of glycosylation type Ic: report of a Japanese case.

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases affecting N-linked glycosylation pathways with variable clinical presentations characterized by psychomotor retardation, seizures, ataxia and hypotonia. CDG-Ic is caused by mutation in the ALG6 gene encoding alpha-1,3-glucosyltransferase. We present a 9-year-old girl diagnosed as having CDG-Ic. She developed severe psychomotor retardation, epileptic seizures, muscle hypotonia, strabismus and some dysmorphic features without inverted nipples or fat pads. She showed a fluctuating serum transaminase level with or without some infection, and a characteristically low level of antithrombin III. MR imaging of the brain at age 2years demonstrated the lower limit of normal myelination, mild atrophy of the cerebrum, and mild hypoplasia of the brainstem and cerebellum. The patient exhibited a CDG type I pattern of serum transferrin on isoelectric focusing and mass spectrometric profiling. Sequence analysis of the ALG6 gene showed two heterozygous mutations, c.998C>T (A333V) and c.1061C>T (P354L). The patient was diagnosed as having CDG-Ic with a novel mutation, making her the first Japanese case. It was suggested that the severe psychomotor retardation in the patient was due to the existence of multiple mutant ALG6 alleles.

Mass spectrometry of apolipoprotein C-III, a simple analytical method for mucin-type O-glycosylation and its application to an autosomal recessive cutis laxa type-2 (ARCL2) patient.

Apolipoprotein C-III (apoCIII) is a small glycoprotein with a single mucin-type core-1 oligosaccharide and is analyzed by isoelectric focusing (IEF) for the diagnosis of genetic defects in O-glycan biosynthesis such as congenital disorders of glycosylation. In the present study, mass spectrometry of apoCIII, after a simple procedure for sample preparation using a small amount of serum, was demonstrated to be a reliable alternative to IEF. It allows reproducible glycan profiling and detection of unglycosylated species. This method was applied to an autosomal recessive cutis laxa type-2 patient and demonstrated decreased site occupancy by O-glycosylation.

Transferrin mutations at the glycosylation site complicate diagnosis of congenital disorders of glycosylation type I.

Congenital disorders of glycosylation (CDG) form a group of metabolic disorders caused by deficient glycosylation of proteins and/or lipids. Isoelectric focusing (IEF) of serum transferrin is the most common screening method to detect abnormalities of protein N-glycosylation. On the basis of the IEF profile, patients can be grouped into CDG type I or CDG type II. Several protein variants of transferrin are known that result in a shift in isoelectric point (pI). In some cases, these protein variants co-migrate with transferrin glycoforms, which complicates interpretation. In two patients with abnormal serum transferrin IEF profiles, neuraminidase digestion and subsequent IEF showed profiles suggestive of the diagnosis of CDG type I. Mass spectrometry of tryptic peptides of immunopurified transferrin, however, revealed a novel mutation at the N-glycan attachment site. In case 1, a peptide with mutation p.Asn630Thr in the 2nd glycosylation site was identified, resulting in an additional band at disialotransferrin position on IEF. After neuraminidase digestion, a single band was found at the asialotransferrin position, indistinguishable from CDG type I patients. In case 2, a peptide with mutation p.Asn432His was found. These results show the use of mass spectrometry of transferrin peptides in the diagnostic track of CDG type I.

Dissociation profile of protonated fucosyl glycopeptides and quantitation of fucosylation levels of glycoproteins by mass spectrometry.

Mass spectrometry of glycopeptides is an efficient strategy for profiling glycans at specific sites in glycoproteins. To assess the reliability of this method for determining the fucosylation levels of glycoproteins, we conducted mass spectrometry of fucosylated glycopeptides from transferrin and haptoglobin. The biantennary glycans containing antenna alpha1,3/4 fucose or alpha1,6 core fucose showed different fragmentation behaviors in collision-induced dissociation of protonated glycopeptides. Stability was dependent on peptide backbone sequences. The major dissociation, cleavage of the GlcNAcbeta1-->2Man linkage of antenna, was evident at a slightly lower activation energy for the core fucosylated species, while the linkage of alpha1,6 core fucose was more stable than that of antenna alpha1,3/4 fucose. However, these fragmentations were induced only with sufficient loading of activation energy. The quantitation of fucosylated glycans by mass spectrometry of glycopeptides, without collisional activation, was thus justified. The fucosylation levels calculated from the signal intensities in electrospray (nanospray) ionization and ultraviolet matrix-assisted laser desorption/ionization mass spectra were essentially the same. The mass spectrometric profiling of glycopeptides from transferrin of congenital disorders of glycosylation (CDG-Ia and CDG-IIc) patients demonstrated that the elevation or reduction of fucosylation in pathological conditions can be reliably determined by MS of glycopeptides.