Quality of life in children with erythropoietic protoporphyria: a case-control study.

Erythropoietic protoporphyria (EPP) is an inherited metabolic disease that causes painful phototoxic reactions, starting in childhood. Studies have shown a reduced quality of life (QoL) in adults with EPP, however, data on children with the disease are lacking. Since treatment for EPP is currently not registered for children, knowledge about their QoL is of crucial importance. In this prospective, case-control study, we included children from the Netherlands and Belgium diagnosed with EPP and matched to healthy controls. Previously collected EPP quality of life (EPP-QoL) data from matched adults with EPP were used. QoL scores, utilizing the Pediatric Quality of Life Inventory (PedsQL) and the disease-specific EPP-QoL, were collected. Scores range from 0 to 100, with higher scores indicating a higher QoL. Non-parametric tests were used to compare groups. A total of 15 cases, 13 matched healthy control children, and 15 matched adults with EPP were included. Children with EPP exhibited lower median scores in the PedsQL in both physical (cases: 87.5 (interquartile range [IQR] 77.7-96.1), controls: 99.2 [IQR 94.9-100.0], p = 0.03) and social (cases: 77.5 [IQR 69.4-86.3], controls: 97.5 [IQR 78.8-100.0], p = 0.04) domains compared to healthy children, although these differences were not statistically significant after correcting for multiple testing. The overall median EPP-QoL score for children was similar to adults with EPP (children: 44.4 [IQR 25.0-54.2], adults: 45.8 [IQR 25.7-68.1], p = 0.68). However, within the EPP-QoL subdomain on QoL, children were found to have significantly lower median scores (children: 16.7 [IQR 0.0-33.3], adults: 33.3 [IQR 33.3-62.5], p < 0.01). In conclusion, children with EPP experience a reduced QoL compared to both healthy children and adults with EPP. Ensuring treatment availability for this patient group is crucial for improving their QoL. We advocate the inclusion of children in safety and efficacy studies, to ensure availability of treatment in the future.

A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

Glycosphingolipids in congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders.

Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome.

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

"Hide and seek": Misleading transferrin variants in PMM2-CDG complicate diagnostics.

PURPOSE: Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism. Despite the availability of next-generation sequencing techniques and advanced methods for evaluation of glycosylation, CDG screening mainly relies on the analysis of serum transferrin (Tf) by isoelectric focusing, HPLC or capillary electrophoresis. The main pitfall of this screening method is the presence of Tf protein variants within the general population. Although reports describe the role of Tf variants leading to falsely abnormal results, their significance in confounding diagnosis in patients with CDG has not been documented so far. Here, we describe two PMM2-CDG cases, in which Tf variants complicated the diagnostic. EXPERIMENTAL DESIGN: Glycosylation investigations included classical screening techniques (capillary electrophoresis, isoelectric focusing and HPLC of Tf) and various confirmation techniques (two-dimensional electrophoresis, western blot, N-glycome, UPLC-FLR/QTOF MS with Rapifluor). Tf variants were highlighted following neuraminidase treatment. Sequencing of PMM2 was performed. RESULTS: In both patients, Tf screening pointed to CDG-II, while second-line analyses pointed to CDG-I. Tf variants were found in both patients, explaining these discrepancies. PMM2 causative variants were identified in both patients. CONCLUSION AND CLINICAL RELEVANCE: We suggest that a neuraminidase treatment should be performed when a typical CDG Tf pattern is found upon initial screening analysis.

Beyond genetics: Deciphering the impact of missense variants in CAD deficiency.

CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.

Pyruvate and uridine rescue the metabolic profile of OXPHOS dysfunction.

INTRODUCTION: Primary mitochondrial diseases (PMD) are a large, heterogeneous group of genetic disorders affecting mitochondrial function, mostly by disrupting the oxidative phosphorylation (OXPHOS) system. Understanding the cellular metabolic re-wiring occurring in PMD is crucial for the development of novel diagnostic tools and treatments, as PMD are often complex to diagnose and most of them currently have no effective therapy. OBJECTIVES: To characterize the cellular metabolic consequences of OXPHOS dysfunction and based on the metabolic signature, to design new diagnostic and therapeutic strategies. METHODS: In vitro assays were performed in skin-derived fibroblasts obtained from patients with diverse PMD and validated in pharmacological models of OXPHOS dysfunction. Proliferation was assessed using the Incucyte technology. Steady-state glucose and glutamine tracing studies were performed with LC-MS quantification of cellular metabolites. The therapeutic potential of nutritional supplements was evaluated by assessing their effect on proliferation and on the metabolomics profile. Successful therapies were then tested in a in vivo lethal rotenone model in zebrafish. RESULTS: OXPHOS dysfunction has a unique metabolic signature linked to an NAD+/NADH imbalance including depletion of TCA intermediates and aspartate, and increased levels of glycerol-3-phosphate. Supplementation with pyruvate and uridine fully rescues this altered metabolic profile and the subsequent proliferation deficit. Additionally, in zebrafish, the same nutritional treatment increases the survival after rotenone exposure. CONCLUSIONS: Our findings reinforce the importance of the NAD+/NADH imbalance following OXPHOS dysfunction in PMD and open the door to new diagnostic and therapeutic tools for PMD.

CAMLG-CDG: a novel congenital disorder of glycosylation linked to defective membrane trafficking.

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.

Genotype-Phenotype Correlations in PMM2-CDG.

PMM2-CDG is a rare disease, causing hypoglycosylation of multiple proteins, hence preventing full functionality. So far, no direct genotype-phenotype correlations have been identified. We carried out a retrospective cohort study on 26 PMM2-CDG patients. We collected the identified genotype, as well as continuous variables indicating the disease severity (based on Nijmegen Pediatric CDG Rating Score or NPCRS) and dichotomous variables reflecting the patients' phenotype. The phenotypic effects of patients' genotype were studied using non-parametric and Chi-Square tests. Seventeen different pathogenic variants have been studied. Variants with zero enzyme activity had no significant impact on the Nijmegen score. Pathogenic variants involving the stabilization/folding domain have a significantly lower total NPCRS (p = 0.017): presence of the p.Cys241Ser mutation had a significantly lower subscore 1,3 and NPCRS (p = 0.04) and thus result in a less severe phenotype. On the other hand, variants involving the dimerization domain, p.Pro113Leu and p.Phe119Leu, resulted in a significantly higher NPCRS score (p = 0.002), which indicates a worse clinical course. These concepts give a better insight in the phenotypic prognosis of PMM2-CDG, according to their molecular base.

Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

Expanding the phenotypic spectrum of FINCA (fibrosis, neurodegeneration, and cerebral angiomatosis) syndrome beyond infancy.

Fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA, MIM#618278) is a rare clinical condition caused by bi-allelic variants in NHL repeat containing protein 2 (NHLRC2, MIM*618277). Pulmonary disease may be the presenting sign and the few patients reported so far, all deceased in early infancy. Exome sequencing was performed on patients with childhood interstitial lung disease (chILD) and additional neurological features. The chILD-EU register database and an in-house database were searched for patients with NHLRC2 variants and clinical features overlapping FINCA syndrome. Six patients from three families were identified with bi-allelic variants in NHLRC2. Two of these children died before the age of two while four others survived until childhood. Interstitial lung disease was pronounced in almost all patients during infancy and stabilized over the course of the disease with neurodevelopmental delay (NDD) evolving as the key clinical finding. We expand the phenotype of FINCA syndrome to a multisystem disorder with variable severity. FINCA syndrome should also be considered in patients beyond infancy with NDD and a history of distinct interstitial lung disease. Managing patients in registers for rare diseases helps identifying new diagnostic entities and advancing care for these patients.

SLC37A4-CDG: Second patient.

Recently, a disorder caused by the heterozygous de novo c.1267C>T (p.R423*) substitution in SLC37A4 has been described. This causes mislocalization of the glucose-6-phosphate transporter to the Golgi leading to a congenital disorder of glycosylation type II (SLC37A4-CDG). Only one patient has been reported showing liver disease that improved with age and mild dysmorphism. Here we report the second patient with a type II CDG caused by the same heterozygous de novo c.1267C>T (p.R423*) mutation thereby confirming the pathogenicity of this variant and expanding the clinical picture with type 1 diabetes, severe scoliosis, and membranoproliferative glomerulonephritis. Additional clinical and biochemical data provide further insight into the mechanism and prognosis of SLC37A4-CDG.

International consensus guidelines for phosphoglucomutase 1 deficiency (PGM1-CDG): Diagnosis, follow-up, and management.

Phosphoglucomutase 1 (PGM1) deficiency is a rare genetic disorder that affects glycogen metabolism, glycolysis, and protein glycosylation. Previously known as GSD XIV, it was recently reclassified as a congenital disorder of glycosylation, PGM1-CDG. PGM1-CDG usually manifests as a multisystem disease. Most patients present as infants with cleft palate, liver function abnormalities and hypoglycemia, but some patients present in adulthood with isolated muscle involvement. Some patients develop life-threatening cardiomyopathy. Unlike most other CDG, PGM1-CDG has an effective treatment option, d-galactose, which has been shown to improve many of the patients' symptoms. Therefore, early diagnosis and initiation of treatment for PGM1-CDG patients are crucial decisions. In this article, our group of international experts suggests diagnostic, follow-up, and management guidelines for PGM1-CDG. These guidelines are based on the best available evidence-based data and experts' opinions aiming to provide a practical resource for health care providers to facilitate successful diagnosis and optimal management of PGM1-CDG patients.

Expanding the clinical and genetic spectrum of CAD deficiency: an epileptic encephalopathy treatable with uridine supplementation.

PURPOSE: Biallelic CAD variants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. METHODS: Retrospective case series of 20 patients. RESULTS: Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. CONCLUSION: We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

Glycogen storage disease type VI: clinical course and molecular background.

Glycogen storage disease type VI (GSD-VI; also known as Hers disease, liver phosphorylase deficiency) is caused by mutations in the gene coding for glycogen phosphorylase (PYGL) leading to a defect in the degradation of glycogen. Since there are only about 40 patients described in literature, our knowledge about the course of the disease is limited. In order to evaluate the long-term outcome of patients with GSD-VI, an observational retrospective case study of six patients was performed at the University Children's Hospital Zurich. The introduction of small, frequent meals as well as cornstarch has led to normal growth in all patients and to normalization of liver transaminases in most patients. After starting the dietary regimen, there were no signs of hypoglycemia. However, three of six patients showed persistent elevation of triglycerides. Further, we identified four novel pathogenic PYGL mutations and describe here their highly variable impact on phosphorylase function.Conclusions: After establishing the diagnosis, dietary treatment led to metabolic stability and to prevention of hypoglycemia. Molecular genetics added important information for the understanding of the clinical variability in this disease. While outcome was overall excellent in all patients, half of the patients showed persistent hypertriglyceridemia even after initiating treatment.What is Known:• Glycogen storage disease type VI (GSD-VI) is a metabolic disorder causing a defect in glycogen degradation. Dietary treatment normally leads to metabolic stability and prevention of hypoglycemia.• However, our knowledge about the natural course of patients with GSD-VI is limited.What is New:• While outcome was overall excellent in all patients, half of the patients showed persistent hypertriglyceridemia even after initiating treatment.• Molecular genetics added important information for the understanding of the clinical variability in this disease.

Clinical and Molecular Characterization of Classical-Like Ehlers-Danlos Syndrome Due to a Novel TNXB Variant.

The Ehlers-Danlos syndromes (EDS) constitute a clinically and genetically heterogeneous group of connective tissue disorders. Tenascin X (TNX) deficiency is a rare type of EDS, defined as classical-like EDS (clEDS), since it phenotypically resembles the classical form of EDS, though lacking atrophic scarring. Although most patients display a well-defined phenotype, the diagnosis of TNX-deficiency is often delayed or overlooked. Here, we described an additional patient with clEDS due to a homozygous null-mutation in the TNXB gene. A review of the literature was performed, summarizing the most important and distinctive clinical signs of this disorder. Characterization of the cellular phenotype demonstrated a distinct organization of the extracellular matrix (ECM), whereby clEDS distinguishes itself from most other EDS subtypes by normal deposition of fibronectin in the ECM and a normal organization of the α5ß1 integrin.

RINT1 Bi-allelic Variations Cause Infantile-Onset Recurrent Acute Liver Failure and Skeletal Abnormalities.

Pediatric acute liver failure (ALF) is life threatening with genetic, immunologic, and environmental etiologies. Approximately half of all cases remain unexplained. Recurrent ALF (RALF) in infants describes repeated episodes of severe liver injury with recovery of hepatic function between crises. We describe bi-allelic RINT1 alterations as the cause of a multisystem disorder including RALF and skeletal abnormalities. Three unrelated individuals with RALF onset ≤3 years of age have splice alterations at the same position (c.1333+1G>A or G>T) in trans with a missense (p.Ala368Thr or p.Leu370Pro) or in-frame deletion (p.Val618_Lys619del) in RINT1. ALF episodes are concomitant with fever/infection and not all individuals have complete normalization of liver function testing between episodes. Liver biopsies revealed nonspecific liver damage including fibrosis, steatosis, or mild increases in Kupffer cells. Skeletal imaging revealed abnormalities affecting the vertebrae and pelvis. Dermal fibroblasts showed splice-variant mediated skipping of exon 9 leading to an out-of-frame product and nonsense-mediated transcript decay. Fibroblasts also revealed decreased RINT1 protein, abnormal Golgi morphology, and impaired autophagic flux compared to control. RINT1 interacts with NBAS, recently implicated in RALF, and UVRAG, to facilitate Golgi-to-ER retrograde vesicle transport. During nutrient depletion or infection, Golgi-to-ER transport is suppressed and autophagy is promoted through UVRAG regulation by mTOR. Aberrant autophagy has been associated with the development of similar skeletal abnormalities and also with liver disease, suggesting that disruption of these RINT1 functions may explain the liver and skeletal findings. Clarifying the pathomechanism underlying this gene-disease relationship may inform therapeutic opportunities.

Mutations in MAGT1 lead to a glycosylation disorder with a variable phenotype.

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. We identified two patients with defective serum transferrin glycosylation and mutations in the MAGT1 gene. These patients present with a phenotype that is mainly characterized by intellectual and developmental disability. MAGT1 has been described to be a subunit of the oligosaccharyltransferase (OST) complex and more specifically of the STT3B complex. However, it was also claimed that MAGT1 is a magnesium (Mg2+) transporter. So far, patients with mutations in MAGT1 were linked to a primary immunodeficiency, characterized by chronic EBV infections attributed to a Mg2+ homeostasis defect (XMEN). We compared the clinical and cellular phenotype of our two patients to that of an XMEN patient that we recently identified. All three patients have an N-glycosylation defect, as was shown by the study of different substrates, such as GLUT1 and SHBG, demonstrating that the posttranslational glycosylation carried out by the STT3B complex is dysfunctional in all three patients. Moreover, MAGT1 deficiency is associated with an enhanced expression of TUSC3, the homolog protein of MAGT1, pointing toward a compensatory mechanism. Hence, we delineate MAGT1-CDG as a disorder associated with two different clinical phenotypes caused by defects in glycosylation.