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.

Neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in The Netherlands: the importance of enzyme analysis to ascertain true MCAD deficiency.

The outcome was determined of population-wide neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using tandem mass spectrometry (MS/MS) in The Netherlands, between October 2003 and September 2005. Prospective population-wide neonatal screening for MCAD deficiency was performed in the northern part of The Netherlands. In newborns with blood octanoylcarnitine (C(8:0)) concentrations > or =0.3 micromol/L, clinical and laboratory follow-up was initiated, including MCAD enzymatic measurements which played a decisive role. In a 2-year period, 66 216 newborns were investigated for MCAD deficiency and follow-up was initiated in 28 newborns. True-positives (n = 14) were identified based upon MCAD enzyme activity <50%, measured with hexanoyl-CoA as substrate. The observed prevalence of MCAD deficiency was 1/6600 (95% CI: 1/4100-1/17 400). In addition to an elevated C(8:0) concentration, a C(8:0)/C(10:0) molar ratio >5.0 turned out to differentiate between false-positives and true-positives. Measurement of MCAD activity using phenylpropionyl-CoA as a substrate further discriminated between newborns with MCAD deficiency and so-called mild MCAD deficiency. To summarize, neonatal screening for MCAD deficiency in the northern part of The Netherlands resulted in the predicted number of affected newborns. Measurement of MCAD activity in leukocytes or lymphocytes using phenylpropionyl-CoA as a substrate can be regarded as the gold standard to diagnose MCAD deficiency upon initial positive screening test results.

[Short-chain acyl-CoA dehydrogenase deficiency (SCADD): relatively high prevalence in the Netherlands and strongly variable fenotype; neonatal screening not indicated]. / De vetzuuroxidatiestoornis 'short chain'-acyl-CoA-dehydrogenase- deficiëntie: relatief hoge prevalentie en sterk wisselend fenotype; neonatale screening niet geïndiceerd.

OBJECTIVE: To describe the clinical, genetic, and biochemical characteristics of short-chain acyl-CoA dehydrogenase deficiency (SCADD), a clinically heterogeneous metabolic disorder for which neonates are screened for in parts of the United States and Australia. To explore the genotype-phenotype relation and to discuss neonatal screening for SCADD. DESIGN: Retrospective study of 31 Dutch SCADD patients and 8 SCADD relatives. METHOD: Patients and relatives were included ifbiochemical SCADD characteristics (increased C4-carnitine and/or ethylmalonic acid) were present in combination with a mutation and/or the c.511C>T or c.625G>A variant on each SCAD-encoding (ACADS) allele. The patients were subdivided into 3 genotype groups: mutation/mutation, mutation/variant and variant/variant group. RESULTS: A birth prevalence for SCADD of at least 1:50,000 was calculated. Most patients presented before the age of 3 years, mainly with developmental delay, epilepsy, behavioural disturbances and/or hypoglycaemia. The ACADS genotype showed a statistically significant association with biochemical, but not with clinical characteristics. In total 7 out of 8 SCADD relatives were free of symptoms. In 5 of the 31 patients, of whom 2 had severe symptoms, a second diagnosis was made which might explain the symptoms. CONCLUSION: SCADD was far more common than had previously been assumed and clinical symptoms in SCADD were non-specific, often transient or absent and not correlated with specific ACADS genotypes. SCADD does not meet major neonatal screening criteria and is therefore not suited for inclusion in neonatal screening programmes.

The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Serotonergic perturbations in dystonia disorders-a systematic review.

Dystonia is a hyperkinetic movement disorder characterized by sustained or intermittent muscle contractions. Emerging data describe high prevalences of non-motor symptoms, including psychiatric co-morbidity, as part of the phenotype of dystonia. Basal ganglia serotonin and serotonin-dopamine interactions gain attention, as imbalances are known to be involved in extrapyramidal movement and psychiatric disorders. We systematically reviewed the literature for human and animal studies relating to serotonin and its role in dystonia. An association between dystonia and the serotonergic system was reported with decreased levels of 5-hydroxyindolacetic acid, the main metabolite of serotonin. A relation between dystonia and drugs affecting the serotonergic system was described in 89 cases in 49 papers. Psychiatric co-morbidity was frequently described, but likely underestimated as it was not systematically examined. Currently, there are no good (pharmaco)therapeutic options for most forms of dystonia or associated non-motor symptoms. Further research using selective serotonergic drugs in appropriate models of dystonia is required to establish the role of the serotonergic system in dystonia and to guide us to new therapeutic strategies.

A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS.

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method is demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.

Identification of a novel mutation (867delA) in the glucose-6-phosphatase gene in two siblings with glycogen storage disease type Ia with different phenotypes.

We identified a novel mutation (867delA) in the glucose-6-phosphatase gene of two siblings with glycogen storage disease type Ia. Although both siblings share the same mutations, their phenotype regarding adult height and hepatomegaly differs. In glycogen storage disease type Ia, substantial heterogeneity in phenotype is observed. So far, no evidence for a clear genotype-phenotype correlation has been found. Hum Mutat 15:381, 2000.

Measurement of short-chain acyl-CoA dehydrogenase (SCAD) in cultured skin fibroblasts with hexanoyl-CoA as a competitive inhibitor to eliminate the contribution of medium-chain acyl-CoA dehydrogenase.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency has so far been reported in only very few patients. This is due, in part, to the problems involved in measuring the activity of SCAD unequivocally. The main reason for this difficulty is that butyryl-CoA, the substrate preferably used for SCAD activity measurements, is also dehydrogenated by medium-chain acyl-CoA dehydrogenase (MCAD). Elimination of this contribution can be achieved by means of immune precipitation with a specific MCAD antibody. We now describe a relatively straightforward assay based on the use of gas chromatography/mass spectrometry for detection. The contribution of MCAD to overall butyryl-CoA dehydrogenation was eliminated by adding excess hexanoyl-CoA to the assay medium. The validity of the method developed was checked by SCAD-activity measurements in fibroblasts from an established SCAD-deficient patient.

Determination of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts using mass spectrometry.

Medium chain acyl-CoA dehydrogenase deficiency, a defect of mitochondrial beta-oxidation, is one of the most frequently occurring among inborn errors of metabolism. We describe a rapid and sensitive gas chromatographic/mass spectrometric method allowing reliable assessment of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts. We investigated MCAD activity in three presumed medium chain acyl-CoA dehydrogenase deficient (MCADD) patients and 10 control subjects. The medium chain acyl-CoA dehydrogenase activity determined in three patients was 1.0 +/- 0.4 nmol.min-1.mg-1 protein (mean +/- SD; range: 0.6-1.4) and in controls it was 2.8 +/- 1.0 nmol.min-1.mg-1 protein (mean +/- SD; range: 1.6-4.4).

[Deficiency of the fatty-acid oxidising enzyme medium-chain acyl-CoA dehydrogenase (MCAD) in an adult, detected during a neonatal screening programme]. / Defïciëntie van het vetzuuroxidatie-enzym middenketen-acyl-coënzym-A-dehydrogenase (MCAD) bij een volwassene, opgespoord tijdens een proefproject voor neonatale screening]

In a trial running since October 2003 in the Dutch provinces of Friesland, Groningen, Drenthe and Overijssel neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency has been added to the regular newborn screening programme for phenylketonuria, congenital hypothyroidism and adrenogenital syndrome. One of the questions to be answered by this trial is the cause of the strong variation in clinical expression of the disorder. Underdiagnosing is an important factor in this phenomenon, as shown by the data of a family of which the case histories of the two oldest children were discussed in this journal in 1965. Both children died at a very young age. Recently, MCAD deficiency was diagnosed in the youngest child of this family, now a 34-year-old woman. This family history illustrates the variable clinical expression of MCAD deficiency, which can cause death but can also run a milder or even subclinical course. Moreover, this family history shows that the underdiagnosis of MCAD deficiency in deceased children may be a cause of the apparently limited clinical detection rate of this disease, for which a simple treatment consisting of life-style and dietary measures is available after diagnosis.

Muscular glycogen storage diseases without increased glycogen content on histopathological examination.

Histopathological findings of muscle biopsies from five patients with two different muscular glycogen storage diseases (mGSD) were presented. From these investigations it emerged that the yield of histopathology in mGSD is low. In only one of five patients histopathological findings gave a clue towards diagnosis. It can be concluded that non-specific findings or even normal appearance of a muscle biopsy does not exclude mGSD.

Pompe disease: current state of treatment modalities and animal models.

Pompe disease is a rare autosomal recessive lysosomal storage disease caused by deficiency of acid-alpha-glucosidase (GAA). This deficiency results in glycogen accumulation in the lysosomes, leading to lysosomal swelling, cellular damage and organ dysfunction. In early-onset patients (the classical infantile form and juvenile form) this glycogen accumulation leads to death. The only therapy clinically available is enzyme replacement therapy, which compensates for the missing enzyme by i.v. administration of recombinant produced enzyme. The development of clinically relevant animal models gained more insight in the disease and allowed evaluation of recombinant enzyme therapy. Several therapies are currently under investigation for Pompe disease, including gene therapy. This review gives an overview of the available knockout mouse models, of the in vitro and in vivo studies performed using recombinant produced enzyme. Furthermore, it describes current therapeutic approaches for Pompe disease as well as experimental therapies like gene correction therapy.

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion.

Partial hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley-Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and hypoxanthine excretion in urine, and decreased activity of hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT). However, normal uric acid concentrations in blood and uric acid excretions in urine have been reported. Here, a boy is presented with normal development and suffering from recurrent attacks of acute renal failure with slightly to clearly increased urinary uric acid excretion. Between these attacks, episodes of elevated urinary excretion of uric acid were observed with normal blood concentrations of uric acid and normal urinary excretion of hypoxanthine. HGPRT activity in erythrocytes, leukocytes, and fibroblasts was found to be strongly decreased. This case shows that not only normal blood uric acid but also normal urinary hypoxanthine concentrations do not exclude the diagnosis of partial HGPRT deficiency.

Type IIIb glycogen storage disease associated with end-stage cirrhosis and hepatocellular carcinoma. The Liver Transplant Group.

Type III glycogen storage disease (GSD) is a disorder of carbohydrate metabolism caused by a deficiency of debranching enzyme. Different subtypes with different clinical pictures have been recognized. During childhood and early adulthood, the symptoms generally regress, and normal adulthood appears possible in most patients without symptoms or signs of cirrhosis. We report on an adult patient with GSD who developed endstage cirrhosis and a small hepatocellular carcinoma. She had GSD subtype IIIb, i.e., there were no signs of cardiomyopathy, myopathy, or neuropathy. She underwent a successful transplantation, representing the first case treated this way for this indication to our knowledge, and she is doing well after 1 year. Debranching enzyme activity was absent both in the liver and in the leukocytes before transplantation. The debranching enzyme activity remained absent in the leukocytes after transplantation. We conclude that patients with GSD type III may develop end-stage cirrhosis and hepatocellular carcinoma and therefore need hepatological follow-up during adulthood.

Glycogen storage disease type Ia: recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flow chart.

UNLABELLED: We studied the glucose-6-phosphatase (G6Pase) gene of 30 unrelated glycogen storage disease type Ia (GSD Ia) patients using single strand conformational polymorphism (SSCP) prior to automated sequencing of exons revealing an aberrant SSCP pattern. In all patients we could identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure. A total of 14 different mutations were identified. R83C (16/60), 158delC (12/60), Q347X (7/60), R170X (6/60) and deltaF327 (4/60) were found most frequently. Nine other mutations accounted for the other 15 mutant alleles. Two DNA-based prenatal diagnoses were performed successfully. At present, 56 mutations in the G6Pase gene have been reported in 300 unrelated GSD Ia patients and an overview of these mutations is presented. Evidence for a clear genotype-phenotype correlation could be established neither from our data nor from those in the literature. With increased knowledge about the genetic basis of GSD Ia and GSD Ib and the high detection rate of mutations, it is our opinion that the diagnoses GSD Ia and GSD Ib can usually be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays in liver tissue obtained by biopsy. A newly developed flowchart for the diagnosis of GSD I is presented. CONCLUSION: Increased knowledge of the genetic basis of glycogen storage disease type I provides a DNA-based diagnosis, prenatal DNA-based diagnosis in chorionic villus samples and carrier detection.

Glycogen storage disease type Ia: four novel mutations (175delGG, R170X, G266V and V338F) identified. Mutations in brief no. 220. Online.

Deficient activity of glucose-6-phosphatase (G6Pase) causes glycogen storage disease type Ia (GSD Ia). We analysed the G6Pase gene of 16 GSD Ia patients using single strand conformation polymorphism (SSCP) analysis prior to automated sequencing of exon(s) revealing an aberrant SSCP pattern. In all GSD Ia patients we were able to identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure to identify mutations. Four novel mutations (175delGG, R170X, G266V and V338F) were identified.