The challenge of CDG diagnosis.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases that currently includes some 130 different types. CDG diagnosis is a challenge, not only because of this large number but also because of the huge clinical heterogeneity even within a number of CDG. In addition, the classical screening test, serum transferrin isoelectrofocusing, is only positive in about 60% of CDG, and can even become negative in some CDG particularly in PMM2-CDG, the most frequent N-glycosylation defect. In order to facilitate CDG diagnosis, we hereby provide some practical tools: (1) a list of clinical features strongly suggestive of a distinctive CDG; (2) a table of clinical, biochemical and laboratory findings reported in CDG, arranged per organ/system; (3) an overview of the affected organs/systems in each CDG; and (4) a diagnostic decision tree in face of a patient with a suspicion of CDG. Most important is to keep in mind a CDG in any unexplained syndrome, in particular when there is neurological involvement. This mini-review enumerates clinical and biochemical hallmarks of these diseases and the biochemical and genetic testing available, and provides an updated list and information on identified CDG. The main aim is to act as a CDG diagnosis simplified guide for healthcare professionals and, additionally, as an awareness and lobbying tool to help in the effectiveness and promptness of CDG diagnosis.

Infantile Alexander Disease with Late Onset Infantile Spasms and Hypsarrhythmia.

Alexander disease (AxD) is a rare autosomal dominant leukodystrophy with three clinical subtypes: infantile, juvenile and adult. Forms differ by age of symptoms occurrence and the clinical presentation. Although recent data suggest considering only two subtypes: type I (infantile onset with lesions extending to the cerebral hemispheres); type II (adult onset with primary involvement of subtentorial structures). Dominant mutations in the glial fibrillary acidic protein (GFAP) gene in AxD cause dysfunction of astrocytes (a type III intermediate filament). The authors discuss the clinical picture of a boy with infantile form of AxD confirmed by the presence of de novo heterozygous mutation c.236G>A in the GFAP gene and without striking symptoms such as macrocephaly and with exceptional late-onset epileptic spasms with hypsarrhyth- mia on electroencephalogram (EEG).

Unique presentation of cutis laxa with Leigh-like syndrome due to ECHS1 deficiency.

Clinical finding of cutis laxa, characterized by wrinkled, redundant, sagging, nonelastic skin, is of growing significance due to its occurrence in several different inborn errors of metabolism (IEM). Metabolic cutis laxa results from Menkes syndrome, caused by a defect in the ATPase copper transporting alpha (ATP7A) gene; congenital disorders of glycosylation due to mutations in subunit 7 of the component of oligomeric Golgi (COG7)-congenital disorders of glycosylation (CDG) complex; combined disorder of N- and O-linked glycosylation, due to mutations in ATPase H+ transporting V0 subunit a2 (ATP6VOA2) gene; pyrroline-5-carboxylate reductase 1 deficiency; pyrroline-5-carboxylate synthase deficiency; macrocephaly, alopecia, cutis laxa, and scoliosis (MACS) syndrome, due to Ras and Rab interactor 2 (RIN2) mutations; transaldolase deficiency caused by mutations in the transaldolase 1 (TALDO1) gene; Gerodermia osteodysplastica due to mutations in the golgin, RAB6-interacting (GORAB or SCYL1BP1) gene; and mitogen-activated pathway (MAP) kinase defects, caused by mutations in several genes [protein tyrosine phosphatase, non-receptor-type 11 (PTPN11), RAF, NF, HRas proto-oncogene, GTPase (HRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), MEK1/2, KRAS proto-oncogene, GTPase (KRAS), SOS Ras/Rho guanine nucleotide exchange factor 2 (SOS2), leucine rich repeat scaffold protein (SHOC2), NRAS proto-oncogene, GTPase (NRAS), and Raf-1 proto-oncogene, serine/threonine kinase (RAF1)], which regulate the Ras-MAPK cascade. Here, we further expand the list of inborn errors of metabolism associated with cutis laxa by describing the clinical presentation of a 17-month-old girl with Leigh-like syndrome due to enoyl coenzyme A hydratase, short chain, 1, mitochondria (ECHS1) deficiency, a mitochondrial matrix enzyme that catalyzes the second step of the beta-oxidation spiral of fatty acids and plays an important role in amino acid catabolism, particularly valine.

Thrombotic complications in patients with PMM2-CDG.

Many proteins regulating coagulation, including factor IX, factor XI, Antithrombin-III, Protein C and Protein S are deficient or decreased in activity in congenital disorders of glycosylation (CDG). Because of the imbalance of coagulation and anticoagulation factors, some patients develop acute vascular events, such as thrombosis. Identifying patients with increased risk for thrombotic events could prevent serious complications and even mortality. We performed a systematic review on patients diagnosed with the most common CDG form; PMM2-CDG, reported between 1990 and 2012 in medical literature. We also evaluated our PMM2-CDG patient-cohort of 15 patients. In total, based on the availability of comprehensive clinical descriptions, 100 patients were included in the study. Patients with and without thrombotic events were compared based on the alterations of the following glycosylated coagulation and anticoagulation factors: Antithrombin-III, Protein C, Protein S, factors IX and XI. We also assessed the global hemostasis, family history and provoking events. In the group of 100 PMM2-CDG patients 14 had suffered a venous or arterial thrombotic event. Low activity of several anticoagulation factors correlated with thrombotic events. Relatively high factor IX and XI activities were not associated with thrombosis. Prolonged PT and aPTT did not seem to protect against thrombosis in patients. Surgical procedures were frequently associated with thrombotic events. Based on the association of thrombosis and surgery in PMM2-CDG we advise to avoid elective surgical procedures in PMM2-CDG patients. Easily preventable risk factors like immobility should be treated with regular physiotherapy. We suggest a yearly follow-up for Antithrombin-III and Protein C levels and parent education for early thrombotic signs in CDG.

Abnormal fat distribution in PMM2-CDG.

We hypothesize that abnormal fat distribution, a common feature of PMM2-CDG, is associated with abnormal perinatal hormone regulation. We assessed 32 cases with PMM2-CDG, for the comorbidity of hypoglycemia/hyperinsulinism and fat pads. Ninety percent of patients with hypoketotic hypoglycemia and/or hyperinsulinism had abnormal fat distribution, while normoglycemic patients showed this feature in 50% of the cases. This statistically significant difference suggests an etiological role of the insulin receptor in developing abnormal fat distribution in PMM2-CDG.

Clinical and diagnostic approach in unsolved CDG patients with a type 2 transferrin pattern.

Dysmorphic features, multisystem disease, and central nervous system involvement are common symptoms in congenital disorders of glycosylation, including several recently discovered Golgi-related glycosylation defects. In search for discriminative features, we assessed eleven children suspected with a Golgi-related inborn error of glycosylation. We evaluated all genetically unsolved patients, diagnosed with a type 2 transferrin isofocusing pattern in the period of 1999-2009. By combining biochemical results with characteristic clinical symptoms, we used a diagnostic flow chart to approach the underlying defect in patients with congenital disorders of glycosylation-IIx. According to specific symptoms and laboratory results, we initiated additional, targeted biochemical and genetic studies. We found a distinctive spectrum of congenital disorders of glycosylation type 2-associated anomalies including sudden hearing loss, brain malformations, wrinkled skin, and epilepsy in combination with skeletal dysplasia, dilated cardiomyopathy, sudden cardiac arrest, abnormal copper and iron metabolism, and endocrine abnormalities in our patients. One patient with severe cortical malformations and mild skin abnormalities was diagnosed with a known genetic syndrome, due to an ATP6V0A2 defect. Here, we present unique congenital disorders of glycosylation type 2-associated anomalies, including both ATPase-related and unrelated cutis laxa and sensorineural hearing loss, a recently recognized symptom of congenital disorders of glycosylation. Based on our findings, we recommend clinicians to consider congenital disorders of glycosylation in patients with cardiac rhythm disorders, spondylodysplasia and biochemical abnormalities of the copper and iron metabolism even in absence of intellectual disability.

Acid sphingomyelinase (Asm) deficiency patients in The Netherlands and Belgium: disease spectrum and natural course in attenuated patients.

Niemann-Pick disease (NPD) is a neurovisceral lysosomal storage disorder caused by acid sphingomyelinase (ASM) deficiency, which can be categorized as either Niemann-Pick disease type A [NPD-A], with progressive neurological disease and death in early childhood, or as Niemann-Pick disease type B [NPD-B], with a more variable spectrum of manifestations. Enzyme replacement therapy (ERT) with recombinant sphingomyelinase is currently studied as potential treatment for NPD-B patients. The objective of this study is to characterize the clinical features of patients with ASM deficiency in the Netherlands and Belgium with focus on the natural disease course of NPD-B patients. Prospective and retrospective data on ASM deficient patients were collected in The Netherlands and part of Belgium. Patients with NPD-B that could be followed prospectively were evaluated every 6-12 months for pulmonary function tests, 6 minute walk test (6 MWT), imaging (bone marrow infiltration measured by QCSI, organ volumes by MRI and CT scan of the lungs) and biochemical markers. Twenty-five patients with ASM deficiency were identified (13 males, 12 females, median age 13years, range 1-59 years). Nine patients had died at the time of the study, including four NPD-A patients at the age of 1,1, 2, 3 and five NPDB patents at the age of 5, 6, 43, 56 and 60 years. There was a high prevalence of homozygosity and compound heterozygosity for the common p.Arg608del mutation in 43% and 19% of NPD-B patients, respectively. In NPD-B patients, thrombocytopenia was present in most, while anemia and leucopenia were less common (33% and 6 % respectively). HDL cholesterol was reduced in most patients. Pulmonary disease was severe in several patients. Follow-up up to 11 years revealed a gradual decrease in platelet count. Detailed investigations in 6 NPD-B patients with follow-up in 4 patients revealed remarkable stable disease parameters up to 6 years, with some decline in pulmonary function and 6 MWT. Bone marrow fat fractions were decreased, indicating the presence of storage macrophages. Lung involvement was not related to the extent of visceromegaly, cytopenia or bone marrow involvement. In conclusion, in NPD-B patients pulmonary disease is the most debilitating feature. Disease manifestations are mostly stable in attenuated patients. Bone marrow infiltration is a less prominent feature of the disease.

Glycosylation defects underlying fetal alcohol spectrum disorder: a novel pathogenetic model. "When the wine goes in, strange things come out" - S.T. Coleridge, The Piccolomini.

Fetal alcohol spectrum disorder (FASD) is an umbrella term used to describe the craniofacial dysmorphic features, malformations, and disturbances in growth, neurodevelopment and behavior occurring in individuals prenatally exposed to alcohol. Fetal alcohol syndrome (FAS) represents the severe end of this spectrum. Many pathophysiological mechanisms have hitherto been proposed to account for the disrupted growth and morphogenesis seen in FAS. These include impaired cholesterol-modification of the Sonic hedgehog morphogen, retinoic acid deficiency, lipoperoxidative damage due to alcohol-induced reactive oxygen species combined with reduced antioxidant defences, and malfunctioning cell adhesion molecules. In this report, we propose a completely novel concept regarding the pathogenesis of FAS. Based on our observation that transferrin isoelectric focusing (TIEF) - the most widely used screening tool for congenital disorders of glycosylation (CDG) - was transiently abnormal in a newborn with FAS and a confirmed maternal history of gestational alcohol abuse, we came to believe that FAS exemplifies a congenital disorder of glycosylation secondary to alcohol-inflicted disruption of (N-linked) protein glycosylation. Various pieces of evidence were found in the literature to substantiate this hypothesis. This observation implies, among others, that one might need to consider the possibility of maternal alcohol consumption in newborns with transient glycosylation abnormalities. We also present an integrated pathophysiological model of FAS, which incorporates all existing theories mentioned above as well as our novel concept. This model highlights the pivotal role of disrupted isoprenoid metabolism in the origination of FAS.

Patient reported outcomes for phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG): listening to what matters for the patients and health professionals.

BACKGROUND: Congenital disorders of glycosylation (CDG) are a growing group of rare genetic disorders. The most common CDG is phosphomannomutase 2 (PMM2)-CDG which often has a severe clinical presentation and life-limiting consequences. There are no approved therapies for this condition. Also, there are no validated disease-specific quality of life (QoL) scales to assess the heterogeneous clinical burden of PMM2-CDG which presents a challenge for the assessment of the disease severity and the impact of a certain treatment on the course of the disease. AIM AND METHODS: This study aimed to identify the most impactful clinical signs and symptoms of PMM2-CDG, and specific patient and observer reported outcome measures (PROMs and ObsROMs, respectively) that can adequately measure such impact on patients' QoL. The most burdensome signs and symptoms were identified through input from the CDG community using a survey targeting PMM2-CDG families and experts, followed by family interviews to understand the real burden of these symptoms in daily life. The list of signs and symptoms was then verified and refined by patient representatives and medical experts in the field. Finally, a literature search for PROMs and ObsROMs used in other rare or common diseases with similar signs and symptoms to those of PMM2-CDG was performed. RESULTS: Twenty-four signs/symptoms were identified as the most impactful throughout PMM2-CDG patients' lifetime. We found 239 articles that included tools to measure those community-selected PMM2-CDG symptoms. Among them, we identified 80 QoL scales that address those signs and symptoms and, subsequently, their psychometric quality was analysed. These scales could be applied directly to the PMM2-CDG population or adapted to create the first PMM2-CDG-specific QoL questionnaire. CONCLUSION: Identifying the impactful clinical manifestations of PMM2-CDG, along with the collection of PROMs/ObsROMs assessing QoL using a creative and community-centric methodology are the first step towards the development of a new, tailored, and specific PMM2-CDG QoL questionnaire. These findings can be used to fill a gap in PMM2-CDG clinical development. Importantly, this methodology is transferable to other CDG and rare diseases with multiple signs and symptoms.

Successful use of albuterol in a patient with central core disease and mitochondrial dysfunction.

Albuterol, a selective beta-adrenergic agonist, has been used experimentally in combination with exercise therapy in a few inherited neuromuscular disorders to increase muscle strength and muscle volume . We report on a 9-year-old boy with central core disease and mitochondrial dysfunction due to compound heterozygous RYR1 mutations receiving albuterol treatment for 1 year. Throughout the period of albuterol administration, the patient underwent an aerobic exercise regime of training sessions three times a week that lasted 20 min each. No side effects of albuterol use were seen. Significant clinical progress, including self care, sitting up, raising arms above the shoulders, independent feeding, and better speech and writing were observed compared with minimal development of these abilities in the previous years on physiotherapy. Improved forced expiratory volume in 1 s (FEV1) score was detected and increased muscle strength was noted: progress was measured using various functional tests and assessment scales. The only complication observed was a mild progression of the joint contractures, possibly due to an unbalance between the flexor and extensor musculature. In general, in this pilot study in a complex case of metabolic myopathy our patient has shown promising results following albuterol treatment and aerobic exercise therapy.

Substrate deprivation therapy in juvenile Sandhoff disease.

Substrate deprivation therapy has been successfully applied in a number of lysosomal storage diseases, such as Gaucher disease. So far only limited experience is available in Sandhoff disease. We initiated substrate deprivation therapy in one male patient, who initially presented at the age of 3.5 years with epilepsy and regression in motor skills and speech development. Juvenile Sandhoff disease was diagnosed on the basis of a decreased hexosaminidase activity in leukocytes and a homozygous HEXB gene mutation. After the epilepsy was controlled, the clinical course remained stable for years, defined by a mild proximal myopathy and stable mental retardation. At 14 years of age the patient experienced a second episode with progressively worsening general condition with diminishing muscle power and progressive ataxia. Treatment was started with the N-alkylated imino sugar miglustat, inhibiting the glucosylceramide synthase, an essential enzyme for the synthesis of glycosphingolipids. Diarrhoea was treated with lactose restriction. We performed detailed biochemical investigations, motor and mental development analysis, brain imaging, organ function studies and quality of life score prior to and at different time points after start of the treatment. Two years after the initiation of therapy the patient has a stable neurological picture without further regression in his motor development, ataxia or intelligence. There is a subjective improvement in the fine motor skills and walking up the stairs but no change in the quality of life score. Under treatment with miglustat the clinical course in our patient with Sandhoff disease did not further deteriorate.

Prediction of outcome in isolated methylmalonic acidurias: combined use of clinical and biochemical parameters.

Objectives Isolated methylmalonic acidurias (MMAurias) are caused by deficiency of methylmalonyl-CoA mutase or by defects in the synthesis of its cofactor 5'-deoxyadenosylcobalamin. The aim of this study was to evaluate which parameters best predicted the long-term outcome. Methods Standardized questionnaires were sent to 20 European metabolic centres asking for age at diagnosis, birth decade, diagnostic work-up, cobalamin responsiveness, enzymatic subgroup (mut(0), mut(-), cblA, cblB) and different aspects of long-term outcome. Results 273 patients were included. Neonatal onset of the disease was associated with increased mortality rate, high frequency of developmental delay, and severe handicap. Cobalamin non-responsive patients with neonatal onset born in the 1970s and 1980s had a particularly poor outcome. A more favourable outcome was found in patients with late onset of symptoms, especially when cobalamin responsive or classified as mut(-). Prevention of neonatal crises in pre-symptomatically diagnosed newborns was identified as a protective factor concerning handicap. Chronic renal failure manifested earlier in mut(0) patients than in other enzymatic subgroups. Conclusion Outcome in MMAurias is best predicted by the enzymatic subgroup, cobalamin responsiveness, age at onset and birth decade. The prognosis is still unfavourable in patients with neonatal metabolic crises and non-responsiveness to cobalamin, in particular mut(0) patients.

Decreased bone density and treatment in patients with autosomal recessive cutis laxa.

AIM: Due to the occasional association pathological fractures and osteoporosis we evaluated four patients with cutis laxa syndrome for skeletal anomalies. PATIENT/METHODS: We prospectively evaluated four patients, a male and a female child and a brother-sister sib pair, with dysmorphic features, growth delay, joint anomalies, psychomotor retardation and congenital cutis laxa. The clinical features and the family history were suggestive for autosomal recessive cutis laxa syndrome type II, partially overlapping with geroderma osteodysplastica. Skeletal survey, sequential bone density measurements, endocrine and metabolic investigations were performed including N- and O-linked glycosylation analysis. ATP6V0A2 and FBLN5 mutations were ruled out in all patients. RESULTS: All children were diagnosed with significantly decreased bone density, especially in the lumbar spine, including spontaneous vertebral and rib fractures in three children. Following 24 months of bisphosphonate treatment a total restitution of bone density was observed in three cases and no relapse was detected in the 2-year follow-up period. A spontaneous improvement was found in one female during puberty. CONCLUSION: Bone disease might occur early in the course in autosomal recessive cutis laxa syndrome. We report on a significant clinical improvement and stabilization in our patients following bisphosphonate therapy. We suggest early, systemic evaluation and follow up of bone density in all children presenting with inherited cutis laxa.

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.

Normal biochemical analysis of the oxidative phosphorylation (OXPHOS) system in a child with POLG mutations: a cautionary note.

We report a 5-year-old child carrying polymerase gamma (POLG1) mutations, but strikingly normal oxidative phosphorylation analysis in muscle, fibroblasts and liver. Mutations in POLG1 have so far been described in children with severe combined oxidative phosphorylation (OXPHOS) deficiencies and with the classical Alpers-Huttenlocher syndrome. The patient presented with a delayed psychomotor development and ataxia during the first two years of life. From the third year of life he developed epilepsy and regression in development, together with symptoms of visual impairment and sensorineuronal deafness. Cerebrospinal fluid showed elevated lactic acid and protein concentrations. An elder brother had died due to combined OXPHOS deficiencies. Despite the clinical similarity with the elder brother, except for liver involvement, the OXPHOS system analysis in a frozen muscle biopsy was normal. For this reason a fresh muscle biopsy was performed, which has the advantage of the possibility of measuring the substrate oxidation rates and ATP production, part of the mitochondrial energy-generating system (MEGS). During the same session, biopsies of liver and fibroblasts were taken. These three tissues showed normal measurements of the MEGS capacity. Based on the phenotype of Alpers-Huttenlocher syndrome in the elder brother, we decided to screen the POLG1 gene. Mutation analysis showed compound heterozygosity with two known mutations, A467T and G848S. The normal MEGS capacity in this patient expands the already existing complexity and heterogeneity of the childhood POLG1 patients and, on the basis of the high frequency of POLG1 mutations in childhood, warrants a liberal strategy with respect to mutation analysis.

Congenital disorder of glycosylation type Ix: review of clinical spectrum and diagnostic steps.

Congenital disorder of glycosylation type I (CDG I) represent a rapidly growing group of inherited multisystem disorders with 13 genetically established subtypes (CDG Ia to CDG Im), and a high number of biochemically unresolved cases (CDG Ix). Further diagnostic effort and prognosis counselling are very challenging in these children. In the current study, we reviewed the clinical records of 10 CDG Ix patients and compared the data with 13 CDG Ix patients published in the literature in search for specific symptoms to create clinical subgroups. The most frequent findings were rather nonspecific, including developmental delay and axial hypotonia. Several features were found that are uncommon in CDG syndrome, such as elevated creatine kinase or arthrogryposis. Distinct ophthalmological abnormalities were observed including optic nerve atrophy, cataract and glaucoma. Two subgroups could be established: one with a pure neurological presentation and the other with a neurological-multivisceral form. The first group had a significantly better prognosis. The unique presentation of microcephaly, seizures, ascites, hepatomegaly, nephrotic syndrome and severe developmental delay was observed in one child diagnosed with CDG Ik. Establishing clinical subgroups and increasing the number of patients within the subgroups may lead the way towards the genetic defect in children with a so far unsolved type of the congenital disorders of glycosylation. Raising awareness for less common, non-CDG specific clinical features such as congenital joint contractures, movement disorders or ophthalmological anomalies will encourage clinicians to think of CDG in its more unusual presentation. Clinical grouping also helps to determine the prognosis and provide better counselling for the families.

Diagnostic work-up and management of patients with isolated methylmalonic acidurias in European metabolic centres.

The long-term outcome of patients with methylmalonic aciduria (MMA) is still uncertain due to a high frequency of complications such as chronic renal failure and metabolic stroke. The understanding of this disease is hampered by a huge variation in the management of these patients. The major aim of this study was to evaluate the current practice in different European metabolic centres. A standardized questionnaire was sent to 20 metabolic centres asking for standard procedures for confirmation of diagnosis, testing cobalamin responsiveness, dietary treatment, pharmacotherapy, and biochemical and clinical monitoring. Sixteen of 20 metabolic centres (80%) returned questionnaires on 183 patients: 89 of the patients were classified as mut(0), 36 as mut(-), 13 as cblA, 7 as cblB, and 38 as cblA/B. (1) Confirmation of diagnosis: All centres investigate enzyme activity by propionate fixation in fibroblasts; six centres also perform mutation analysis. (2) Cobalamin response: Ten centres follow standardized protocols showing large variations. A reliable exclusion of nonspecific effects has not yet been achieved by these protocols. (3) Long-term treatment: In cobalamin-responsive patients, most centres use hydroxocobalamin (1-14 mg/week i.m. or 5-20 mg/week orally), while two centres use cyanocobalamin. All cobalamin-nonresponsive patients and most cobalamin-responsive patients are supplemented with L: -carnitine (50-100 mg/kg per day). Fourteen centres use intestinal decontamination by antibiotic therapy. Most centres follow D-A-CH (n = 6) or Dewey (n = 4) recommendations for protein requirements. Fourteen centres regularly use precursor-free amino acid supplements. Standardized monitoring protocols are available in seven centres, again showing high variability.