Decreased oxidative phosphorylation and PGAM deficiency in horses suffering from atypical myopathy associated with acquired MADD.

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation. In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls. The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values. Most respiratory chain complex activity in patients was 20-30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses. General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised. This study expands the diagnostic possibilities of equine myopathies.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates.

Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering alpha-glucosidases, which are present in granulocytes but not in lymphocytes. Here we show that the application of acarbose in the enzymatic assay employing the artificial substrate 4-methylumbelliferyl-alpha-D: -glucoside (MU-alphaGlc) is insufficient to clearly distinguish patients from healthy individuals in all cases. Also, the ratios of the activities without/with acarbose only marginally discriminated Pompe patients and healthy individuals. By contrast, when the natural substrate glycogen is used, the activity in leukocytes from patients (n = 82) with Pompe disease is at most 17% of the lowest control value. The use of artificial substrate in an assay with isolated lymphocytes instead of total leukocytes is a poor alternative as blood samples older than one day invariably yield lymphocyte preparations that are contaminated with granulocytes. To diagnose Pompe disease in leukocytes we recommend the use of glycogen as substrate in the presence of acarbose. This assay unequivocally excludes Pompe disease. To also exclude pseudo-deficiency of acid alpha-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-alphaGlc substrate plus acarbose or DNA analysis is required.

Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands.

Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo C syndrome) is a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). We performed a clinical study on 29 Dutch MPS IIIC patients and determined causative mutations in the recently identified HGSNAT gene. Psychomotor development was reported to be normal in all patients during the first year of life. First clinical signs were usually noted between 1 and 6 years (mean 3.5 years), and consisted of delayed psychomotor development and behavioral problems. Other symptoms included sleeping and hearing problems, recurrent infections, diarrhoea and epilepsy. Two sisters had attenuated disease and did not have symptoms until the third decade. Mean age of death was 34 years (range 25-48). Molecular analysis revealed mutations in both alleles for all patients except one. Altogether 14 different mutations were found: two splice site mutations, one frame shift mutation due to an insertion, three nonsense mutations and eight missense mutations. Two mutations, p.R344C and p.S518F, were frequent among probands of Dutch origin representing 22.0% and 29.3%, respectively, of the mutant alleles. This study demonstrates that MPS IIIC has a milder course than previously reported and that both severity and clinical course are highly variable even between sibs, complicating prediction of the clinical phenotype for individual patients. A clear phenotype-genotype correlation could not be established, except that the mutations p.G262R and p.S539C were only found in two sisters with late-onset disease and presumably convey a mild phenotype.

Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting.

Pompe disease is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). It presents at any age, with variable rates of progression ranging from a rapidly progressive course, often fatal by one-year of age, to a more slowly, but nevertheless relentlessly progressive course, resulting in significant morbidity and premature mortality. In infants, early initiation of enzyme replacement therapy is needed to gain the maximum therapeutic benefit, underscoring the need for early diagnosis. Several new methods for measuring GAA activity have been developed. The Pompe Disease Diagnostic Working Group met to review data generated using the new methods, and to establish a consensus regarding the application of the methods for the laboratory diagnosis of Pompe disease. Skin fibroblasts and muscle biopsy have traditionally been the samples of choice for measuring GAA activity. However, new methods using blood samples are rapidly becoming adopted because of their speed and convenience. Measuring GAA activity in blood samples should be performed under acidic conditions (pH 3.8-4.0), using up to 2 mM of the synthetic substrate 4-methylumbelliferyl-alpha-D-glucoside or glycogen (50 mg/mL), in the presence of acarbose (3-9 microM) to inhibit the isoenzyme maltase-glucoamylase. The activity of a reference enzyme should also be measured to confirm the quality of the sample. A second test should be done to support the diagnosis of Pompe disease until a program for external quality assurance and proficiency testing of the enzymatic diagnosis in blood is established.

Sanfilippo syndrome: a mini-review.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is an autosomal recessive disorder, caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of the glycosaminoglycan heparan sulfate. Based on the enzyme deficiency, four different subtypes, MPS IIIA, B, C, and D, are recognized. The genes encoding these four enzymes have been characterized and various mutations have been reported. The probable diagnosis of all MPS III subtypes is based on increased concentration of heparan sulfate in the urine. Enzymatic assays in leukocytes and/or fibroblasts confirm the diagnosis and allow for discrimination between the different subtypes of the disease. The clinical course of MPS III can be divided into three phases. In the first phase, which usually starts between 1 and 4 years of age, a developmental delay becomes apparent after an initial normal development during the first 1-2 years of life. The second phase generally starts around 3-4 years and is characterized by severe behavioural problems and progressive mental deterioration ultimately leading to severe dementia. In the third and final stage, behavioural problems slowly disappear, but motor retardation with swallowing difficulties and spasticity emerge. Patients usually die at the end of the second or beginning of the third decade of life, although survival into the fourth decade has been reported. Although currently no effective therapy is yet available for MPS III, several promising developments raise hope that therapeutic interventions, halting the devastating mental and behavioural deterioration, might be feasible in the near future.

Juvenile-onset neuronal ceroid lipofuscinosis with infantile CLN1 mutation and palmitoyl-protein thioesterase deficiency.

Accurate diagnosis, especially in progressive hereditary diseases, is essential for the treatment and genetic counseling of the patient and the family. Neuronal ceroid lipofuscinoses (NCL) are amongst the most common groups of neurodegenerative diseases. Infantile, juvenile, and adult-onset types with multiple genotype-phenotype associations have been described. A fluorimetric enzyme assay for palmitoyl protein thioesterase (PPT) from leukocytes and fibroblasts has been previously developed to confirm the diagnosis of infantile NCL. We describe a patient with juvenile-onset NCL phenotype with a new CLN1 mutation and deficient PPT activity. Over 40 different mutations have been found in patients with PPT deficiency, indicating that screening for known mutations is not an efficient way to diagnose this disorder. Therefore, PPT enzyme analysis should precede mutation analysis in suspected PPT deficiency, particularly in patients with granular osmiophilic deposits (GROD) or in patients who have negative ultrastructural data. The use of enzyme assay led to the diagnosis of this patient with juvenile-onset Finnish variant NCL with PPT deficiency, and we expect that greater awareness of the utility of the enzymatic assay may lead to identification of other similar cases awaiting a definitive diagnosis.

Localisation of a gene for mucopolysaccharidosis IIIC to the pericentromeric region of chromosome 8.

Mucopolysaccharidosis type IIIC (MPS IIIC, or Sanfilippo syndrome C) is a rare lysosomal storage disorder caused by a deficiency of acetyl-coenzyme A:alpha-glucosaminide-N-acetyltransferase. Patients develop progressive neuropsychiatric problems, mental retardation, hearing loss, and relatively minor visceral manifestations. The pattern of transmission is consistent with an autosomal recessive mode of inheritance. The aim of this study was to find a locus for MPS IIIC using a homozygosity mapping approach. A genomewide scan was performed on DNA from 27 affected individuals and 17 of their unaffected relatives. Additional patients were recruited, and DNA was obtained from a total of 44 affected individuals and 18 unaffected family members from 31 families from 10 countries. A working candidate interval was defined by looking for excess homozygosity in patients compared with their relatives. Additional markers were genotyped in regions of interest. Linkage analysis was performed to support the informal analysis. Inspection of the genomewide scan data showed apparent excess homozygosity in patients compared with their relatives for markers on chromosome 8. Additional genotyping identified 15 consecutive markers (from D8S1051 to D8S2332) in an 8.3 cM interval for which the genotypes of affected siblings were identical in state. A maximum multipoint lod score of 10.61 was found at marker D8S519. A locus for MPS IIIC maps to an 8.3 cM (16 Mbp) interval in the pericentromeric region of chromosome 8.

[From gene to disease; from CLN1, CLN2 and CLN3 to neuronal ceroid lipofuscinosis]. / Van gen naar ziekte; van Clni, CLN2 en CLN3 naar neuronale ceroidlipofuscinose.

The neuronal ceroid lipofuscinoses (NCL) are worldwide the most common lysosomal storage disorders of childhood. Clinical features often include progressive visual impairment, seizures, psychomotor deterioration, dementia, and premature death. Most NCL cases are caused by mutations in the CLN1, CLN2 and CLN3 genes, which play an essential role in lysosomal protein degradation. Laboratory diagnostics for a patient suspected of NCL should start with enzyme analysis in the case of INCL and LINCL and investigation of lymphocyte vacuolisation for JNCL. Diagnosis at the protein level is not available for JNCL, but CLN3 mutation analysis is possible. The carrier status of healthy relatives in families with known mutations in either CLN1, CLN2, CLN3 or CLN6 can be determined with certainty by mutation analysis.

Genomic organization of the human phosphomannose isomerase (MPI) gene and mutation analysis in patients with congenital disorders of glycosylation type Ib (CDG-Ib).

CDG-Ib is the "gastro-intestinal" type of the congenital disorders of glycosylation (CDG) and a potentially treatable disorder. It has been described in patients presenting with congenital hepatic fibrosis and protein losing enteropathy. The symptoms result from hypoglycosylation of serum- and other glycoproteins. CDG-Ib is caused by a deficiency of mannose-6-phosphate isomerase (synonym: phosphomannose isomerase, EC 5.3.1.8), due to mutations in the MPI gene. We determined the genomic structure of the MPI gene in order to simplify mutation detection. The gene is composed of 8 exons and spans only 5 kb. Eight (7 novel) different mutations were found in seven patients with a confirmed phosphomannose isomerase deficiency, analyzed in the context of this study: six missense mutations, a splice mutation and one insertion. In the last, the mutation resulted in an unstable transcript, and was hardly detectable at the mRNA level. This emphasizes the importance of mutation analysis at the genomic DNA level.

Is the perinatal lethal form of Gaucher disease more common than classic type 2 Gaucher disease?

In recent years there has been increased recognition of a severe perinatal lethal form of Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase. We previously reported a case of severe type 2 Gaucher disease which was seen in a medical center in Rotterdam and now present three new cases from two other families seen at the same center. Mutational analyses of these cases revealed two novel mutations, H311R and V398F, located in exons 8 and 9, respectively. The identification of four cases of lethal type 2 Gaucher disease in a single center seems to be a function of increased awareness of this phenotype, rather than of geographic clustering. The actual incidence of lethal type 2 Gaucher disease may be underestimated, as many cases may have been misclassified as collodion babies or hydrops of unknown cause.

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity.

Sphingolipid activator proteins are small glycoproteins required for the degradation of sphingolipids by specific lysosomal hydrolases. Four of them, called saposins, are encoded by the prosaposin gene, the product of which is proteolytically cleaved into the four mature saposin proteins (saposins A, B, C, D). One of these, saposin B, is necessary in the hydrolysis of sulphatide by arylsulphatase A where it presents the solubilised substrate to the enzyme. As an alternative to arylsulphatase A deficiency, deficiency of saposin B causes metachromatic leukodystrophy. We identified a previously undescribed mutation (N215K) in the prosaposin gene of a patient with metachromatic leukodystrophy but with normal arylsulphatase A activity and elevated sulphatide in urine. The mutation involves a highly conserved amino acidic residue and abolishes the only N-glycosylation site of saposin B.

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?

Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological symptoms. Both sibs are homozygous for the E325K mutation, the same genotype that was found in the most severe form of alpha-NAGA deficiency presenting as infantile neuroaxonal dystrophy. Thus, at the age of 7 years the same genotype of alpha-NAGA may present as a 'non-disease' (present healthy case) and can be associated with the vegetative state (the first two patients described with alpha-NAGA deficiency). The clinical heterogeneity among the 11 known individuals with alpha-NAGA deficiency is extreme, with a 'non-disease' (two cases) and infantile neuroaxonal dystrophy (two cases) at the opposite sides of the clinical spectrum. The broad spectrum is completed by a very heterogeneous group of patients with various degrees of epilepsy/behavioural difficulties/psychomotor retardation (four patients) and a mild phenotype in adults without overt neurological manifestations who have angiokeratoma and clear vacuolisation in various cell types (three cases). These observations are difficult to reconcile with a straightforward genotype-phenotype correlation and suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the 11 patients with alpha-NAGA deficiency.

A diagnostic protocol for adult-onset glycogen storage disease type II.

To analyze the diagnostic value of various laboratory tests for the confirmation of adult-onset glycogen storage disease type II (GSD II), we performed a clinical, biochemical, and genetic study of 18 patients with this disease. Measurement of acid alpha-glucosidase (GAA) activity in muscle and histopathologic analysis of muscle tissue appeared to have no additional value when GAA activity in leukocytes was clearly deficient. Our study showed that creatine kinase elevation is a sensitive marker of GSD II. A diagnostic protocol is formulated.

A novel missense mutation in the glycogen branching enzyme gene in a child with myopathy and hepatopathy.

We have identified a novel missense mutation in the gene for glycogen branching enzyme (GBE 1) in a 16-month-old infant with a combination of hepatic and muscular features, an atypical clinical presentation of glycogenosis type IV (GSD IV). The patient was heterozygous for a G-to-A substitution at codon 524 (R524Q), changing an encoded arginine (CGA) to glutamine (CAA), while the GBE1 gene on the other allele was not expressed. This case broadens the spectrum of mutations in patients with GSD IV and confirms the clinical and molecular heterogeneity of this disease.

Prenatal diagnosis of cystic kidney disease with ventriculomegaly: a report of six cases in two related sibships.

In 2 consanguineous relationships, a Cape Verdian man fathered six fetuses (5 male) with fetal ventriculomegaly and echodense fetal kidneys as visualized by ultrasonography between 16 and 32 weeks. During prenatal monitoring, an increased alpha fetoprotein level and abnormal acetylcholinesterase were detected at amniocentesis in 5 of 6 affected fetuses. Chromosomes were normal. Five pregnancies resulted in elective termination; one child was still-born prematurely. Hydrocephalus and cystic disease of the (renal) cortico medullary areas were found. One fetus had polydactyly. The differential diagnosis and prenatal diagnosis of this presumably autosomal recessive syndrome are discussed.

Morquio B syndrome: a primary defect in beta-galactosidase.

Fibroblasts from patients with Morquio B syndrome contain normal numbers of beta-galactosidase molecules with normal turnover but strongly reduced activity per enzyme molecule. Various substrate affinities are abnormal: the Km for methylum belliferyl (MU)-beta-galactoside is 4-10-fold elevated and affinity for keratan sulphate and oligosaccharides, isolated from Morquio B urine, was not detectable. In contrast, these substrate affinities are normal for beta-galactosidase in adult type GM1-gangliosidosis fibroblasts. Cell hybridization studies demonstrate that Morquio B syndrome and infantile and adult type GM1-gangliosidosis belong to the same complementation group. From these results we conclude that Morquio B syndrome is caused by a mutation in the structural gene for beta-galactosidase, which is allelic to the mutations in infantile and adult type GM1-gangliosidosis. Urinary excretion of keratan sulphate and oligosaccharides is abnormal in Morquio B syndrome but normal in adult type GM1-gangliosidosis. The catalytic properties of beta-galactosidase in Morquio B syndrome and GM1-gangliosidosis provide a possible explanation for the distinct clinical manifestations in these disorders.

Different phenotypic expression in relatives with fabry disease caused by a W226X mutation.

Two male relatives with Fabry disease presented striking differences in clinical symptoms and age of onset. The propositus had retarded statural growth and skeletal dysplasia while his nephew suffered mainly from aggravating acroparesthesia and celiac disease. Fabry disease is an X-linked inborn error of glycosphingolipid metabolism resulting from deficient activity of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal A) enzyme. The alpha-Gal A gene is located at Xq22.1. Efforts to establish genotype-phenotype correlations have been limited because most patients have private mutations. In previous clinical studies performed in families with Fabry disease, marked differences in phenotype are described between affected relatives. This family also demonstrates the difficulty in predicting the clinical phenotype in patients and relatives with the same alpha-Gal A mutation. Furthermore, in the absence of a family history, the diagnosis may be easily missed.

Submicroscopic Xpter deletion in a boy with growth and mental retardation caused by a familial t(X;14).

In a 3-year-old boy with short stature, developmental delay, and dry skin, steroid sulphatase deficiency and a submicroscopic terminal deletion of Xp were found. Except for the short stature, no major clinical signs of X-linked recessive chondrodysplasia punctata could be observed. His mother had lowered steroid sulphatase activity compatible with carriership for X-linked ichthyosis and a submicroscopic translocation (X;14)(p22.31;p11.1). This finding combined with a normal amplification of exons 1, 5, and 10 of the STS gene from propositus' DNA suggested a breakpoint upstream of the STS gene. The submicroscopic maternal translocation had important implications for genetic counseling. This case report illustrates that contiguous gene syndrome related to the Xpter region may have an atypical clinical presentation and the usefulness of combined clinical, biochemical, molecular, and fluorescence in situ hybridization analysis.

Asymptomatic and late-onset ornithine transcarbamylase deficiency caused by a A208T mutation: clinical, biochemical and DNA analyses in a four-generation family.

We describe a 4-generation family in which a previously healthy 10-year-old boy died of late-onset ornithine transcarbamylase (OTC) deficiency. Pedigree analysis and allopurinol loading tests in female relatives were not informative. A missense mutation (A208T) in the OTC gene was detected in the deceased patient and in several clinically healthy male and female relatives, the oldest male being 97 years old. OTC deficiency was established in autopsy liver tissue of the propositus and liver biopsy samples of his sister, mother, and a maternal uncle. The males had 4% and 6% residual activity, respectively, the females 58% and 67%, respectively. The observed relation between the mutation and the decreased OTC activity in liver tissue of these subjects suggests that the mutation is a deleterious one. Late-onset, "mild" OTC deficiency can have a fatal or a favorable outcome. The disease can segregate undetected in families.